Materials and methods for the treatment of Duchenne muscular dystrophy

Abstract

To provide materials and methods for treatment of Duchenne muscular dystrophy.SOLUTION: The present application presents materials and methods for treating a patient with Duchenne muscular dystrophy (DMD) both ex vivo and in vivo. In addition, the present application presents materials and methods for editing a dystrophin gene in a cell by genome editing. The present disclosure presents an approach to address the genetic basis of DMD. By using genome engineering tools to create permanent changes in the genome that can restore the dystrophin reading frame and the dystrophin protein activity with as few as a single treatment, the resulting therapy can correct the underlying genetic defect causing the disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field This application presents materials and methods for treating patients with Duchenne muscular dystrophy (DMD) both ex vivo and in vivo. In addition, this application presents materials and methods for editing the dystrophin gene in cells by genome editing.

[0002] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 247,484, filed October 28, 2015, and U.S. Provisional Application No. 62 / 324,064, filed April 18, 2016, both of which are incorporated herein by reference in their entirety.

[0003] Incorporation by Reference of Sequence Listing This application may be downloaded in computer readable form (filename: 160101PCT sequence listing_ST25; 286,928,896 bytes; ASCII text file; 2016 (created October 28, 2013), which is incorporated by reference in its entirety and forms part of this disclosure. [Background technology]

[0004] background Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disorder that occurs in approximately 1 in 4,000 live births in boys. Patients are typically diagnosed by the age of 4 and are wheelchair-bound by the age of 10. Most patients do not live beyond the age of 25 due to heart and / or respiratory failure. Existing treatments are palliative at best. The most common treatment for DMD is steroids, which are used to slow the loss of muscle strength. However, because most DMD patients begin taking steroids at a young age, the treatment delays puberty and further contributes to a decreased quality of life for patients.

[0005] DMD is caused by mutations in the dystrophin gene (X chromosome: 31,117,228-33,344,609 (Genome Reference Consortium: GRCh38 / hg38)). Dystrophin, with a genomic region spanning 2.2 megabases, is the second largest human gene. The dystrophin gene contains 79 exons, which are processed into an 11,000-base pair mRNA, which is translated into a 427-kDa protein. Functionally, dystrophin acts as a linker between actin filaments and the extracellular matrix within muscle fibers. The N-terminus of dystrophin is an actin-binding domain, while the C-terminus interacts with a transmembrane scaffold that anchors muscle fibers to the extracellular matrix. During muscle contraction, dystrophin provides structural support that allows muscle tissue to withstand mechanical forces. DMD is caused by a variety of mutations in the dystrophin gene that result in a premature stop codon, resulting in a truncated dystrophin protein. The truncated dystrophin protein does not contain the C-terminus and therefore cannot provide the structural support necessary to withstand the stress of muscle contraction. As a result, muscle fibers are pulled apart, leading to muscle wasting.

[0006] Becker muscular dystrophy (BMD) is a less severe form of muscular dystrophy compared to DMD. BMD is also caused by mutations in the dystrophin gene, but the mutations in BMD maintain the dystrophin reading frame. The BMD dystrophin protein contains an internal deletion but retains both the N- and C-terminal portions. Thus, the dystrophin protein in BMD is shorter than the wild-type protein, but can still function as a linker between actin filaments and the extracellular matrix. In fact, depending on the size of the internal deletion, BMD patients may only show mild symptoms. As a result, most research efforts focus on converting the severe DMD phenotype into a less severe BMD phenotype.

[0007] Genome engineering refers to strategies and techniques for targeted and specific modification of an organism's genetic information (genome). Genome engineering is a highly active area of ​​research due to its wide range of potential applications, particularly in the areas of human health; correcting genes carrying harmful mutations, for example, exploring gene function. Early techniques developed to insert genes into living cells, such as genetic engineering, were often limited by the random nature of the insertion of new sequences into the genome. New genes were typically placed blindly, sometimes inactivating or disrupting the function of other genes, or even causing severe undesirable effects. Furthermore, these techniques generally did not provide any degree of reproducibility, as there was no guarantee that the new sequences would be inserted into the same location in two different cells. More recent genome engineering strategies, such as ZFN, TALEN, HE, and MegaTAL, allow for the modification of specific regions of DNA, thereby increasing the accuracy of correction or insertion compared to earlier techniques and providing a degree of reproducibility. Despite this, these recent genome engineering strategies have limitations.

[0008] Several studies suggest that genome manipulation is an attractive strategy for treating DMD. One of the earliest approaches involved engineering a mini-dystrophin gene that was less than 4 kb and could be packaged into an adeno-associated virus (AAV) vector. This was demonstrated in a mouse model (Wang, B., J. Li, and X. Xiao, Proc Natl Acad Sci U SA, 2000, 97(25):13714-9) (Watchko, J. et al., Hum Gene Ther, 2002, 13(12):1451-60) and in a dog model (Wang, Z. et al., Mol Ther, 2012, 20(8):1501-7). This is the first gene replacement therapy investigated, and Phase I clinical trials have suggested that there are problems associated with immune responses to non-self synthetic epitopes (Mendell, JR et al., N Engl J Med, 2010, 363(15):1429-37).

[0009] More recently, oligo-mediated exon skipping has been used to restore the reading frame in DMD patients' cells. In this strategy, short oligos block splicing signals found in pre-mRNA, facilitating single-exon skipping. Single-exon skipping allows the transcriptional machinery to bypass premature stop codons and generate proteins with intact N- and C-termini. Phase I / II clinical trials have shown that weekly injections of antisense oligos induce exon skipping and dystrophin-positive fibers (Cirak, S. et al., Lancet, 2011, 378(9791):595-605). However, a major limitation of this type of treatment is that the drug targets pre-mRNA rather than genomic loci, requiring repeated administration throughout the patient's life. Ongoing phase II / III clinical trials are assessing AAV-mediated delivery of exon-skipping oligos for sustained expression, as well as delivery of multiple antisense oligos to facilitate multi-exon skipping strategies. Despite the efforts of researchers and medical professionals worldwide attempting to address DMD, and despite the promise of genome engineering approaches, there remains an urgent need to develop safe and effective treatments for DMD, the most prevalent and debilitating genetic disorder. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Wang, B., J. Li, and X. Xiao, Proc Natl Acad Sci USA, 2000, 97(25):13714–9. [Non-patent document 2] Watchko, J. et al., Hum Gene Ther, 2002, 13(12):1451-60 [Non-patent document 3] Wang, Z. et al., Mol Ther, 2012, 20(8):1501-7 [Non-patent document 3] Mendell, JR et al., N Engl J Med, 2010, 363(15):1429-37 [Non-patent document 4] Cirak, S. et al., Lancet, 2011, 378(9791):595-605 Summary of the Invention [Means for solving the problem]

[0011] Abstract This disclosure presents an approach to address the genetic basis of DMD: using genome engineering tools to create permanent changes to the genome that can restore the dystrophin reading frame and restore dystrophin protein activity in as few as one treatments, the resulting therapy can correct the underlying genetic defect that causes the disease.

[0012] This paper provides an ex vivo and in vivo cellular method for creating permanent changes in genome by genome editing, by deleting, inserting, or replacing (deleting and inserting) one or more exons or splice acceptor or donor site of abnormal intron in dystrophin gene, thereby restoring dystrophin reading frame and restoring dystrophin protein activity, and this ex vivo and in vivo cellular method can be used to treat Duchenne muscular dystrophy (DMD).This paper also provides components, kits and compositions for carrying out this method.Also provided are the cells produced by this method.

[0013] Provided herein is a method for editing a dystrophin gene in a human cell by genome editing, the method comprising introducing one or more deoxyribonucleic acid (DNA) endonucleases into the human cell to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, which result in the permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, thereby restoring the dystrophin reading frame and restoring the activity of the dystrophin protein. The human cell may be a muscle cell or a muscle progenitor cell.

[0014] Also provided herein is an ex vivo method for treating a patient (e.g., a human) with Duchenne muscular dystrophy (DMD), the ex vivo method comprising: i) creating DMD patient-specific induced pluripotent stem cells (iPSCs); ii) editing the iPSCs within or near the dystrophin gene; iii) differentiating the genome-edited iPSCs into Pax7+ muscle progenitor cells; and iv) implanting the Pax7+ muscle progenitor cells into the patient.

[0015] The step of creating patient-specific induced pluripotent stem cells (iPSCs) may include: a) isolating somatic cells from a patient; and b) introducing a set of pluripotency-associated genes into the somatic cells to induce the somatic cells to become pluripotent stem cells. The somatic cells may be fibroblasts. The set of pluripotency-associated genes is one or more genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

[0016] Editing iPSCs within or near the dystrophin gene can include introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSCs to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene that result in a permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0017] The step of differentiating the genome-edited iPSCs into Pax7+ muscle progenitor cells may include contacting the genome-edited iPSCs with a specific media formulation that includes a small molecule drug; overexpression of a transgene; or serum deprivation.

[0018] The step of implanting the Pax7+ muscle progenitor cells into the patient can include implanting the Pax7+ muscle progenitor cells into the patient by local injection into the desired muscle.

[0019] Also provided herein is an in vivo method for treating a patient (e.g., a human) with Duchenne muscular dystrophy (DMD), the method comprising editing the dystrophin gene in the patient's cells. The cells can be muscle cells or muscle progenitor cells.

[0020] Editing dystrophin in the patient's cells may include introducing into the patient's cells one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene that result in a permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, thereby restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0021] The one or more DNA endonucleases may be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr2, Csm3, Csm4, Csm5, Csm6, Cmr1, Csm2, Csm3, Csm4, Csm5, Csm6, Csm7, Csm8, Csm9, Csm10, Csm11, Csm12, Csm13, Csm14, Csm15, Csm16, Csm17, Csm18, Csm1 ...9, Csm10, Csm11, Csm12, Csm11, Csm12, Csm13 Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; homologs thereof, recombinant versions of these naturally occurring molecules, codon-optimized versions thereof, modified versions thereof, and any combination of the foregoing.

[0022] The method may include introducing into the cell one or more polynucleotides encoding one or more DNA endonucleases. The method may include introducing into the cell one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases. The one or more polynucleotides or one or more RNAs may be one or more modified polynucleotides or one or more modified RNAs. The one or more DNA endonucleases may be one or more proteins or polypeptides.

[0023] The method may further comprise introducing one or more guide ribonucleic acids (gRNAs) into the cell. The one or more gRNAs are single-molecule guide RNAs (sgRNAs). The one or more gRNAs or the one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs. The one or more DNA endonucleases can be pre-complexed with the one or more gRNAs or the one or more sgRNAs.

[0024] The method may further comprise the step of introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type dystrophin gene or cDNA. At least a portion of the wild-type dystrophin gene or cDNA may be selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, exon 80, exon 81, exon 82, exon exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the entire dystrophin gene or cDNA.At least a portion of the wild-type dystrophin gene or cDNA may comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, The donor template may comprise exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA. The donor template may be a single-stranded or double-stranded polynucleotide.

[0025] The method may further comprise introducing one or more guide ribonucleic acids (gRNAs) into the cell. The one or more DNA endonucleases may be one or more Cas9 or Cpf1 endonucleases that generate a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), where a first SSB or DSB is cleaved at the 5' locus and a second SSB or DSB is cleaved at the 3' locus, resulting in the permanent deletion or replacement of one or more exons or splice acceptor or donor sites of an aberrant intron within or near the dystrophin gene, thereby restoring the dystrophin reading frame and restoring the activity of the dystrophin protein. One gRNA may create a pair of SSBs or DSBs. One gRNA may contain a spacer sequence that is complementary to the 5' locus, the 3' locus, or a segment between the 5' and 3' loci. The first gRNA may contain a spacer sequence that is complementary to a segment at the 5' locus, and the second gRNA may contain a spacer sequence that is complementary to a segment at the 3' locus.

[0026] The one or more gRNAs may be one or more single-molecule guide RNAs (sgRNAs). The one or more gRNAs or one or more sgRNAs may be one or more modified gRNAs or one or more modified sgRNAs. One or more DNA endonucleases may be pre-complexed with the one or more gRNAs or one or more sgRNAs.

[0027] There may be a deletion of chromosomal DNA between the 5' and 3' loci.

[0028] The deletion may be a deletion of a single exon. The single exon deletion may be a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, or exon 53. The 5' locus may be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. The 3' locus may be proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53. The 5' locus can be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53, and the 3' locus can be proximal to the 3' boundary of such a single exon. Proximal to the boundary of an exon can include splice donors and acceptors around the adjacent intron.

[0029] The deletion may be a multi-exon deletion. The multi-exon deletion may be a deletion of exons 45-53 or exons 45-55. The 5' locus may be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55. The 3' locus may be proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55. The 5' locus may be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55, and the 3' locus may be proximal to these 3' boundaries. Proximal to the exon boundary may include splice donors and acceptors around adjacent introns.

[0030] A replacement of chromosomal DNA may exist between the 5' locus and the 3' locus. The replacement may be a replacement of a single exon. The replacement of a single exon may be a replacement of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5' locus may be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 3' locus can be proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5' locus can be proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70, and the 3' locus can be proximal to these 3' boundaries. Proximal to the exon boundary can include splice donors and acceptors around the adjacent intron or exon.

[0031] The replacement may be a multi-exon replacement. The multi-exon replacement may be a replacement of exons 45-53 or exons 45-55. The 5' locus may be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55. The 3' locus may be proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55. The 5' locus may be proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55, and the 3' locus may be proximal to these 3' boundaries. Proximal to the exon boundary may include splice donors and acceptors around adjacent introns or adjacent exons.

[0032] The method can further include introducing into the cell a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene or cDNA, wherein replacement is by homology-directed repair (HDR).

[0033] At least a portion of the wild-type dystrophin gene or cDNA may be selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, exon 80, exon 81, exon 82, exon exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the entire dystrophin gene or cDNA.At least a portion of the wild-type dystrophin gene or cDNA may comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, The fragments may include exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

[0034] The method may further include introducing into the cell a guide ribonucleic acid (gRNA) and a polynucleotide donor template containing at least a portion of a wild-type dystrophin gene. The one or more DNA endonucleases may be one or more Cas9 or Cpf1 endonucleases that generate a single-strand break (SSB) or double-strand break (DSB) at a locus within or near the dystrophin gene that facilitates the insertion of a new sequence derived from the polynucleotide donor template into chromosomal DNA at the locus, resulting in the permanent insertion or correction of one or more exons or aberrant intron splice acceptor or donor sites within or near the dystrophin gene, restoring the dystrophin reading frame and restoring the activity of the dystrophin protein. The gRNA may include a spacer sequence complementary to a segment of the locus.

[0035] The method may further include introducing into the cell one or more guide ribonucleic acids (gRNAs) and a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene. The one or more DNA endonucleases may be one or more Cas9 or Cpfl endonucleases that generate a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), the first at the 5' locus and the second at the 3' locus, that facilitate insertion of new sequences derived from the polynucleotide donor template into chromosomal DNA between the 5' and 3' loci within or near the dystrophin gene, resulting in permanent insertion or correction of one or more exons or aberrant intron splice acceptor or donor sites, restoring the dystrophin reading frame and restoring dystrophin protein activity.

[0036] One gRNA can create a pair of SSBs or DSBs. One gRNA can contain a spacer sequence complementary to the 5' locus, the 3' locus, or a segment between the 5' and 3' loci. The first gRNA can contain a spacer sequence complementary to a segment at the 5' locus, and the second gRNA can contain a spacer sequence complementary to a segment at the 3' locus.

[0037] The one or more gRNAs may be one or more single-molecule guide RNAs (sgRNAs). The one or more gRNAs or one or more sgRNAs may be one or more modified gRNAs or one or more modified sgRNAs. One or more DNA endonucleases may be pre-complexed with the one or more gRNAs or one or more sgRNAs.

[0038] There may be an insertion between the 5' and 3' loci.

[0039] The insertion can be an insertion of a single exon. The single exon insertion can be an insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. The 5' locus or 3' locus can be proximal to a boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and exon 70. Proximal to an exon boundary can include splice donors and acceptors around adjacent introns or adjacent exons.

[0040] The insertion may be a multi-exon insertion. The multi-exon insertion may be an insertion of exons 45-53 or exons 45-55. The 5' locus or 3' locus may be proximal to a boundary of multiple exons selected from the group consisting of exons 45-53 or exons 45-55. Proximal to an exon boundary may include a splice donor and acceptor around an adjacent intron.

[0041] At least a portion of the wild-type dystrophin gene or cDNA may be selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, exon 80, exon 81, exon 82, exon exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or at least a portion of the entire dystrophin gene or cDNA.At least a portion of the wild-type dystrophin gene or cDNA may comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, The fragments may include exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

[0042] The insertion or correction can be by homology-directed repair (HDR).

[0043] The donor template can be a single-stranded or double-stranded polynucleotide.

[0044] The Cas9 or Cpf1 mRNA, gRNA, and donor template can each be formulated into separate lipid nanoparticles, or all can be co-formulated into lipid nanoparticles.

[0045] Cas9 or Cpf1 mRNA can be formulated into lipid nanoparticles, and both the gRNA and donor template can be delivered to cells by adeno-associated viral (AAV) vectors.

[0046] Cas9 or Cpf1 mRNA can be formulated into lipid nanoparticles, gRNA can be delivered to cells by electroporation, and donor templates can be delivered to cells by adeno-associated virus (AAV) vectors.

[0047] The dystrophin gene may be located on the X chromosome: 31,117,228 to 33,344,609 (Genome Reference Consortium: GRCh38 / hg38).

[0048] Also provided herein are one or more guide ribonucleic acids (gRNAs) for editing the dystrophin gene in cells derived from patients with DMD. The one or more gRNAs and / or sgRNAs may comprise a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 1 to 1,410,472 of the Sequence Listing. The one or more gRNAs may be one or more single-molecule guide RNAs (sgRNAs). The one or more gRNAs or one or more sgRNAs may be one or more modified gRNAs or one or more modified sgRNAs.

[0049] Provided herein are cells modified by the preceding methods to permanently delete or correct one or more exons, or aberrant intron splice acceptor or donor sites, within the dystrophin gene, to restore the dystrophin reading frame and restore the activity of the dystrophin protein. Provided herein are further methods for ameliorating DMD by administering the cells modified by the preceding methods to DMD patients.

[0050] It is understood that the invention described herein is not limited to the examples summarized in this Summary. Various other embodiments are also described and illustrated herein.

[0051] Various aspects of the materials and methods for the treatment of DMD disclosed and described herein can be better understood with reference to the accompanying drawings. [Brief explanation of the drawings]

[0052] [Figure 1A] Figure 1A shows a plasmid (CTx-1) containing a codon-optimized gene for the S. pyogenes Cas9 endonuclease. The CTx-1 plasmid also contains a gRNA scaffold sequence, which includes a 20-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1 to 467,030 in the Sequence Listing, or a 19-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1,410,430 to 1,410,472 in the Sequence Listing.

[0053] [Figure 1B] Figure 1B shows a plasmid (CTx-2) containing a different codon-optimized gene for the S. pyogenes Cas9 endonuclease. The CTx-2 plasmid also contains a gRNA scaffold sequence, which includes a 20-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1 to 467,030 in the Sequence Listing, or a 19-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1,410,430 to 1,410,472 in the Sequence Listing.

[0054] [Figure 1C] Figure 1C shows a plasmid (CTx-3) containing another codon-optimized gene for the S. pyogenes Cas9 endonuclease. The CTx-3 plasmid also contains a gRNA scaffold sequence, which includes a 20-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1 to 467,030 in the Sequence Listing, or a 19-bp spacer sequence from any of the sequences listed in SEQ ID NOS: 1,410,430 to 1,410,472 in the Sequence Listing.

[0055] [Figure 2A] Figure 2A is a diagram of a type II CRISPR / Cas system.

[0056] [Figure 2B] Figure 2B is a diagram of a Type II CRISPR / Cas system.

[0057] [Figure 3A] Figure 3A illustrates the cleavage efficiency of S. pyogenes gRNAs in HEK293T targeting exons 45, 51, and 53 of the dystrophin gene.

[0058] [Figure 3B] Figure 3B illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T targeting exons 55 and 70 of the dystrophin gene.

[0059] [Figure 4A] Figure 4A illustrates the cleavage efficiency of S. pyogenes gRNAs in HEK293T targeting the splice acceptor of exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene.

[0060] [Figure 4B] Figure 4B illustrates the cleavage efficiency of N. meningitides, S. thermophiles, and S. aureus gRNAs in HEK293T targeting the splice acceptor of exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene.

[0061] [Figure 4C]Figure 4C illustrates the cleavage efficiency of Cpf1 gRNA in HEK293T targeting the splice acceptor of exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the dystrophin gene.

[0062] [Figure 5A] Figure 5A-B illustrates the cleavage efficiency and splice acceptor knockout efficiency of S. pyogenes gRNAs in HEK293T targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene. [Figure 5B] Figure 5A-B illustrates the cleavage efficiency and splice acceptor knockout efficiency of S. pyogenes gRNAs in HEK293T targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene.

[0063] [Figure 6] Figure 6 illustrates the cleavage efficiency and splice acceptor knockout efficiency of N. meningitides (NM), S. thermophiles (ST), and S. aureus (SA) gRNAs in HEK293T targeting exons 51, 45, 53, 44, 46, 52, 50, 43, and 55 of the dystrophin gene.

[0064] [Figure 7A] Figure 7A illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T cells, where the gRNA targets the region surrounding exon 52 of the dystrophin gene.

[0065] [Figure 7B] Figure 7B illustrates the cleavage efficiency of S. pyogenes gRNA in HEK293T cells, where the gRNA targets the region surrounding exons 44, 45, and 54 of the dystrophin gene.

[0066] [Figure 8A] Figure 8A illustrates the cleavage efficiency of S. pyogenes gRNA in iPSCs, where the gRNA targets the region surrounding exon 52 of the dystrophin gene.

[0067] [Figure 8B] Figure 8B illustrates the cleavage efficiency of S. pyogenes gRNA in iPSCs, where the gRNA targets the region surrounding exons 44, 45, and 54 of the dystrophin gene.

[0068] [Figure 9] Figure 9 illustrates a comparison of the cleavage efficiency of S. pyogenes gRNAs in HEK293T cells and iPSCs, where the gRNAs target the region surrounding exons 44, 45, 52, and 54 of the dystrophin gene.

[0069] [Figure 10AB] Figures 10A, 10B, and 10C illustrate the clonal analysis of clonal deletion events. [Figure 10C] Figures 10A, 10B, and 10C illustrate the clonal analysis of clonal deletion events.

[0070] [Figure 11] Figures 11A and 11B illustrate Sanger sequencing of the Δ52 clone.

[0071] [Figure 12A] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcription (IVT) gRNA screening. [Figure 12B] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcription (IVT) gRNA screening. [Figure 12C]Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcription (IVT) gRNA screening. [Figure 12D] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcription (IVT) gRNA screening. [Figure 12E] Figures 12A-E illustrate the cleavage efficiency of gRNAs selected by in vitro transcription (IVT) gRNA screening.

[0072] [Figure 13A] Figure 13A illustrates homology-directed repair (HDR) between exons 45 and 55 of the dystrophin gene.

[0073] [Figure 13B] Figure 13B shows PCR confirmation of HDR at the exon 45-55 locus of the dystrophin gene.

[0074] [Figure 14A] Figure 14A shows a three-primer PCR assay.

[0075] [Figure 14B] Figure 14B shows the results from a three-primer PCR assay.

[0076] [Figure 14C] FIG. 14C illustrates data generated from a three-primer PCR assay.

[0077] [Figure 15] Figure 15 illustrates five clones carrying the desired Δ45-55 deletion.

[0078] [Figure 16]Figures 16A-B illustrate the SSEA-4 and TRA-160 staining results of five clones carrying the desired Δ45-55 deletion.

[0079] [Figure 17] Figure 17 shows the expression of the internally deleted dystrophin protein for all five edited clones.

[0080] [Figure 18] FIG. 18 shows myosin heavy chain staining of differentiated clone 56.

[0081] [Figure 19A] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19B] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19C] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19D] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19E] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19F] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19G] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19H] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19I] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19J] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19K] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19L] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19M] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19N] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19O] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19P] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19Q] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19R] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19S] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19T] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19U] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19V] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19W] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19X] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19Y]19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19Z] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19AA] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19BB] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19CC] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19DD] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19EE] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19FF] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19GG] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19HH] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19II] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19JJ] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19KK] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19LL] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19MM] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19NN] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19OO] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19PP] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19QQ] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19RR] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19SS] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19TT] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19UU] 19A-19VV illustrate the results of a large-scale lentiviral screen. [Figure 19VV] 19A-19VV illustrate the results of a large-scale lentiviral screen. DETAILED DESCRIPTION OF THE INVENTION

[0082] Brief description of the sequence listing SEQ ID NOs: 1-467,030 are a list of gRNA 20 bp spacer sequences for targeting the dystrophin gene with S. pyogenes Cas9 endonuclease.

[0083] SEQ ID NOs: 467,031 to 528,196 are a list of gRNA 20 bp spacer sequences for targeting the dystrophin gene with S. aureus Cas9 endonuclease.

[0084] SEQ ID NOs: 528,197 to 553,198 are a list of gRNA 24 bp spacer sequences for targeting the dystrophin gene with S. thermophilus Cas9 endonuclease.

[0085] SEQ ID NOs: 553,199 to 563,911 are a list of gRNA 24 bp spacer sequences for targeting the dystrophin gene with T. denticola Cas9 endonuclease.

[0086] SEQ ID NOs: 563,912 to 627,854 are a list of gRNA 24 bp spacer sequences for targeting the dystrophin gene with N. meningitides Cas9 endonuclease.

[0087] SEQ ID NOs: 627,855 to 1,410,399 are a listing of gRNA 20-24 bp spacer sequences for targeting the dystrophin gene in Acidominoccoccus, Lachnospiraceae, and Franciscella Novicida Cpf1 endonucleases.

[0088] SEQ ID NOs: 1,410,400 to 1,410,402 are N. meningitides List of gRNA 24bp spacer sequences for targeting the dystrophin gene with Cas9 endonuclease.

[0089] SEQ ID NOs: 1,410,403 to 1,410,429 are a list of gRNA 23 bp spacer sequences for targeting the dystrophin gene in Acidominoccoccus, Lachnospiraceae, and Franciscella Novicida Cpf1 endonucleases.

[0090] SEQ ID NOs: 1,410,430 to 1,410,472 are a list of gRNA 19 bp spacer sequences for targeting the dystrophin gene with S. pyogenes Cas9 endonuclease.

[0091] Detailed Description Duchenne muscular dystrophy (DMD)

[0092] DMD is caused by mutations in the dystrophin gene (X chromosome: 31,117,228-33,344,609 (Genome Reference Consortium: GRCh38 / hg38)). Dystrophin, with a genomic region spanning 2.2 megabases, is the second largest human gene. The dystrophin gene contains 79 exons, which are processed into an 11,000-base pair mRNA, which is translated into a 427-kDa protein. Functionally, dystrophin acts as a linker between actin filaments and the extracellular matrix within muscle fibers. The N-terminus of dystrophin is an actin-binding domain, while the C-terminus interacts with a transmembrane scaffold that anchors muscle fibers to the extracellular matrix. During muscle contraction, dystrophin provides structural support, allowing muscle tissue to withstand mechanical forces. DMD is caused by a variety of mutations in the dystrophin gene that result in a premature stop codon, resulting in a truncated dystrophin protein. The truncated dystrophin protein does not contain the C-terminus and therefore cannot provide the structural support necessary to withstand the stress of muscle contraction. As a result, muscle fibers are pulled apart, resulting in muscle wasting.

[0093] treatment

[0094] Presented herein are ex vivo and in vivo cellular methods for creating permanent changes to the genome using genome engineering tools that can restore the dystrophin reading frame and restore the activity of the dystrophin protein. These methods use endonucleases, such as CRISPR / Cas9 nucleases, to permanently delete (excise), insert, or replace (delete and insert) exons within the genomic locus of the dystrophin gene (i.e., mutations within the coding and / or splicing sequences). In this way, the present invention mimics the products created by exon skipping and / or restores the reading frame with a single, minimal treatment (rather than delivering exon skipping oligos for the patient's lifetime). Preclinical studies have been conducted on the expression of the C-terminus of dystrophin by using zinc finger, TALE, and CRISPR / Cas9-based nucleases to make targeted changes to the genome. In one example, a deletion of a large genomic region was performed that is estimated to treat over 60% of patients with DMD.

[0095] This paper provides a method for treating patients with DMD.The example of this method is ex vivo cell-based therapy.For example, create DMD patient-specific iPS cell line.Then, use the materials and methods described herein to correct the chromosomal DNA of these iPS cells.Then, corrected iPSCs are differentiated into Pax7+ muscle progenitor cells.Finally, progenitor cells are transplanted into patients.This ex vivo method has many advantages.

[0096] One advantage of ex vivo cell therapy is the ability to perform comprehensive analysis of therapeutic agents prior to administration. All nuclease-based therapeutic agents exert some level of off-target effects. Performing ex vivo gene correction allows for complete characterization of the corrected cell population prior to implantation. Aspects of the present disclosure include sequencing the entire genome of the corrected cells to ensure that off-target cleavage, if present, is in a genomic location associated with minimal risk to the patient. Furthermore, clonal populations of cells can be isolated prior to implantation.

[0097] Another advantage of ex vivo cell therapy relates to gene correction in iPSCs compared to other primary cell sources. iPSCs are prolific, facilitating the large numbers of cells required for cell-based therapies. Furthermore, iPSCs are an ideal cell type for performing clonal isolation, allowing for precise genomic correction without the risk of viability loss. In contrast, other potential cell types, such as primary myoblasts, only survive for a few passages and are difficult to clonally expand. Patient-specific DMD myoblasts can also be unhealthy due to the lack of dystrophin protein. On the other hand, patient-derived DMD iPSCs do not express dystrophin in this differentiated state and therefore do not display the diseased phenotype. Therefore, manipulation of DMD iPSCs is much easier and may shorten the amount of time required to achieve the desired gene correction.

[0098] An additional advantage of ex vivo cell therapy relates to the implantation of myogenic Pax7+ progenitor cells versus myoblasts. Pax7+ cells are accepted as myogenic satellite cells. Pax7+ progenitor cells are mononuclear cells present at the periphery of multinucleated muscle fibers. In response to injury, progenitor cells divide and fuse with existing fibers. In contrast, myoblasts fuse directly with muscle fibers upon implantation and have minimal proliferative capacity in vivo. Therefore, whereas myoblasts cannot contribute to healing after repeated injury, Pax7+ progenitor cells may function as a reservoir and contribute to muscle healing over the patient's lifetime.

[0099] Another example of such a method is an in vivo-based therapy, in which the materials and methods described herein are used to correct the chromosomal DNA of cells in a patient.

[0100] The advantage of in vivo gene therapy is the ease of generating and administering therapeutic agents. The same therapeutic cocktail may potentially reach a subset (n>1) of the DMD patient population. In contrast, proposed ex vivo cell therapy requires custom-made therapeutic agents to be developed for each patient (n=1). The development of ex vivo cell therapy requires time, which may not be available for certain advanced DMD patients.

[0101] Also provided herein is a cell method for genome editing to edit the dystrophin gene in human cell.For example, cell is isolated from patient or animal.Then, the chromosomal DNA of cell is corrected using material and method as described herein.

[0102] In addition to mutations in coding and splicing sequences, several types of genomic target sites may exist.

[0103] Regulation of transcription and translation involves several different classes of sites that interact with proteins or nucleotides within the cell. DNA-binding sites of transcription factors or other proteins can often be targeted for mutation or deletion to study the site's role, but they can also be targeted to alter gene expression. Sites can be added through direct genome editing by nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The increasing use of genome sequencing, RNA expression, and genome-wide studies of transcription factor binding has increased our ability to identify how sites mediate developmental or temporal gene regulation. These control systems can be direct or involve extensive cooperative regulation that may require the integration of activity from multiple enhancers. Transcription factors typically bind to degenerate DNA sequences 6–12 bp in length. The low level of specificity afforded by individual sites suggests that complex interactions and rules are involved in binding and functional outcomes. Less degenerate binding sites may provide a more convenient means of regulation. Artificial transcription factors can be designed to specify longer sequences with fewer similar sequences in the genome, reducing the potential for off-target cleavage. Any of these types of binding sites can be mutated, deleted, or even created to allow for altered gene regulation or expression (Canver, MC et al., Nature (201 5 years)).

[0104] Another class of gene regulatory regions that possess these characteristics are microRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play a key role in post-transcriptional gene regulation. miRNAs can regulate the expression of 30% of all mammalian protein-coding genes. Specific and potent gene silencing (RNAi) by double-stranded RNA, plus additional small non-coding RNAs, has been discovered (Canver, MC et al., Nature (2015)). Non-coding RNAs important for gene silencing The largest class of miRNAs are miRNAs. In mammals, miRNAs are initially transcribed as long RNA transcripts that can be individual transcription units, parts of protein introns, or other transcripts. The long transcripts, called pri-miRNAs (primary miRNAs), contain imperfectly base-paired hairpin structures. These pri-miRNAs can be cleaved into one or more short pre-miRNAs (precursor miRNAs) by the Microprocessor, a nuclear protein complex that involves Drosha.

[0105] The pre-miRNA is a short stem-loop approximately 70 nucleotides long with a 2-nucleotide 3' overhang that is exported to the 19-25 nucleotide mature miRNA:miRNA * The stable base-pairing small miRNA strand (guide strand) can be loaded into the RNA-induced silencing complex (RISC). * Mature miRNAs (marked with ) can be functional but are typically degraded. Mature miRNAs tether RISC to partially complementary sequence motifs in target mRNAs, primarily found in the 3' untranslated region (UTR), leading to post-transcriptional gene silencing (Bartel, D.P., Cell, 136, 215-233 (2009); Saj, A. and Lai, E.C., Curr Opin Genet Dev, 21, 504-510 (2010)). 11 years)).

[0106] miRNAs may be important in the control of development, differentiation, cell cycle, and proliferation, as well as in virtually every biological pathway in mammals and other multicellular organisms. miRNAs may also be involved in cell cycle control, apoptosis, and stem cell differentiation, hematopoiesis, hypoxia, myogenesis, neurogenesis, insulin secretion, cholesterol metabolism, aging, viral replication, and the immune response.

[0107] While a single miRNA can target hundreds of different mRNA transcripts, an individual transcript can be targeted by many different miRNAs. In the latest version of miRBase (v.21), over 28,645 microRNAs have been annotated. Some miRNAs may be encoded by multiple loci, some of which may be expressed from tandemly co-transcribed clusters. This feature allows for complex regulatory networks involving multiple pathways and feedback control. miRNAs may be an integral part of these feedback and regulatory circuits and may help regulate gene expression by keeping protein production within limits (Herranz, H. and Cohen, SM, Gene Dev, 24, 1339-1344 (2010); Posadas, DM and and Carthew, RW, Curr Opin Genet Dev, 27, 1-6 (2014).

[0108] miRNAs may also be important in many human diseases associated with abnormal miRNA expression. This association supports the importance of miRNA regulatory pathways. Recent miRNA deletion studies have linked miRNAs to the regulation of immune response (Stern-Ginossar, N. et al., Science, 317, 376-381 (2007)).

[0109] miRNAs also have a strong connection with cancer and may play a role in different types of cancer. miRNAs have been found to be downregulated in some tumors. miRNAs may be important in regulating key cancer-related pathways, such as cell cycle control and response to DNA damage, and therefore can be used in diagnosis and targeted in clinical settings. microRNAs can precisely regulate the angiogenesis balance, such that experiments that deplete all microRNAs suppress tumor angiogenesis (Chen, S. et al., Genes Dev, 28, 1054-1067 (2014)).

[0110] As has been shown for protein-coding genes, miRNA genes may also be subject to epigenetic changes that occur with cancer. Many miRNA loci may be associated with CpG islands, which increases the chance of their regulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B., and Lyko, F., Cell Cycle, 6, 1001-1005 (2007)). The majority of studies have focused on chromatin-specific miRNAs. Treatment with miRNA-remodeling drugs has been used to reveal epigenetic silencing of miRNAs.

[0111] In addition to their role in RNA silencing, miRNAs can also activate translation (Posadas, DM and Carthew, RW, Curr Opin Genet Dev 27:1-6 (2014)). Knocking out these sites can result in decreased expression of the target gene, whereas introducing these sites can increase expression.

[0112] Individual miRNAs can be most effectively knocked out by mutating the seed sequence (a 2-8 base microRNA) that may be important for binding specificity. Cleavage within this region, followed by misrepair by NHEJ, can effectively abolish miRNA function by blocking binding to the target site. miRNAs could also be inhibited by specific targeting of the special loop region adjacent to the palindromic sequence. Catalytically inactive Cas9 can also be used to inhibit the expression of shRNAs (Zhao, Y. et al., Sci Rep 4, 3943 (2014)). miRNA targeting In addition to silencing, the binding site can also be targeted and mutated to prevent silencing by the miRNA.

[0113] human cells

[0114] As described and illustrated herein, the primary target for gene editing to improve DMD is human cell.For example, in ex vivo method, human cell can be somatic cell that can be modified by using the described technique and can produce Pax7+ muscle progenitor cell.For example, in in vivo method, human cell can be muscle cell or muscle progenitor cell.

[0115] By performing gene editing in autologous cells that originate from the patient in need thereof, and are therefore already a perfect match for the patient, it is possible to generate cells that can be safely reintroduced into the patient, effectively resulting in a cell population that may be effective in ameliorating one or more clinical conditions associated with the patient's disease.

[0116] Progenitor cells (also referred to herein as stem cells) have the capacity to both proliferate and give rise to more progenitor cells, and to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and subsequently differentiate into one or more mature cell types, while also giving rise to progeny that retain one or more cells with the developmental potential of the parent. The term "stem cell," then, refers to a cell that, under certain circumstances, retains the ability to proliferate, with the ability or potential to differentiate into a more specialized or differentiated phenotype, and, under certain circumstances, without substantially differentiating. In one aspect, the term progenitor or stem cell refers to a general-purpose mother cell whose progeny (progeny) often specialize in different directions by differentiating, for example, by acquiring entirely individual characteristics, as in the ongoing diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from multipotent cells, which are themselves derived from multipotent cells, and so on. Each of these multipotent cells is considered a stem cell, although the range of cell types each can give rise to can vary greatly. Some differentiated cells also have the ability to give rise to cells of greater developmental potential. This ability can be natural or can be artificially induced upon treatment with various factors. In many biological cases, stem cells can also be "multipotent" because they can give rise to progeny of more than one distinct cell type, although this is not required to be "stem."

[0117] Self-renewal may be another important aspect of stem cells. Theoretically, self-renewal can occur through either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter cell retaining the stem cell state and the other daughter cell expressing some other distinct, specific function and phenotype. Alternatively, a portion of stem cells within a population may divide symmetrically into two stem cells, thereby maintaining some stem cells within the population as a whole, while other cells within the population give rise to only differentiated progeny. Generally, "progenitor cells" have a cellular phenotype that is more primitive (i.e., at an earlier stage along the developmental pathway or progression than fully differentiated cells). Progenitor cells also have a pronounced or very high proliferative potential. Progenitor cells may give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and environment in which the cells develop and differentiate.

[0118] In the context of cellular ontogeny, the adjectives "differentiated" or "to differentiate" are relative terms. A "differentiated cell" is one that is further down the developmental pathway than the cell it is being compared to. Thus, stem cells can differentiate into lineage-restricted progenitor cells (such as muscle cell progenitors), which can differentiate into other types of progenitor cells further down the pathway (such as muscle cell precursors), which then differentiate into terminally differentiated cells, such as muscle cells, that may or may not retain the ability to play the characteristic role in a particular tissue type and further proliferate.

[0119] induced pluripotent stem cells

[0120] In some examples, the genetically engineered human cells described herein can be induced pluripotent stem cells (iPSCs). An advantage of using iPSCs is that the cells can be derived from the same subject to which the progenitor cells are administered. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells for administration to the subject (e.g., autologous cells). Because progenitor cells are essentially derived from an autologous source, the risk of engraftment rejection or allergic response can be reduced compared to the use of cells derived from another subject or group of subjects. In addition, the use of iPSCs also eliminates the need for cells obtained from an embryonic source. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.

[0121] Although differentiation is generally irreversible in a physiological context, several methods have recently been developed for reprogramming somatic cells into iPSCs. Exemplary methods are known to those skilled in the art and are outlined herein below.

[0122] The term "reprogramming" refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). In other words, reprogramming refers to a process that drives the differentiation of a cell back toward a less differentiated or more primitive cell type. It should be noted that culturing many primary cells can result in some loss of complete differentiation characteristics. Thus, simply culturing such cells within the term differentiated cells does not render them non-differentiated (e.g., undifferentiated) or pluripotent. The transition from differentiated cells to pluripotency requires a reprogramming stimulus beyond one that results in a partial loss of differentiated characteristics in culture. Reprogrammed cells also possess the characteristic of expansion potential without loss of proliferative potential compared to primary parent cells, which generally have the capacity to divide only a limited number of times in culture.

[0123] The cell to be reprogrammed may be partially differentiated or terminally differentiated prior to reprogramming. Reprogramming encompasses the complete reversal of the differentiated state of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. Reprogramming may encompass the complete or partial reversal of the differentiated state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming may result in the expression of specific genes by the cell, the expression of which further contributes to the reprogramming. In certain examples described herein, reprogramming a differentiated cell (e.g., a somatic cell) may cause the differentiated cell to adopt an undifferentiated state (e.g., the differentiated cell may become an undifferentiated cell). The resulting cell is referred to as a "reprogrammed cell" or "induced pluripotent stem cell (iPSC or iPS cell)."

[0124] Reprogramming can involve altering, e.g., reversing, at least some of the inherited patterns that occur during cell differentiation, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. Reprogramming is distinct from simply maintaining the existing undifferentiated state of an already pluripotent cell, or maintaining the existing less-than-fully differentiated state of an already multipotent cell (e.g., a myogenic stem cell). Reprogramming is also distinct from promoting self-renewal or proliferation of already pluripotent or multipotent cells, although in some instances, the compositions and methods described herein can also be useful for such purposes.

[0125] Many methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Any such method that reprograms somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

[0126] Reprogramming methods for generating pluripotent cells using defined combinations of transcription factors have been described. Direct transduction of Oct4, Sox2, Klf4, and c-Myc can convert mouse somatic cells into ES cell-like cells with expanded developmental potential (see, e.g., Takahashi and Yamanaka, Cell, 126(4):663-76 (2006)). iPSCs resemble ES cells because they restore much of the pluripotency-associated transcriptional circuitry and epigenetic landscape. In addition, mouse iPSCs fulfill all standard assays for pluripotency: specifically, in vitro differentiation into cell types of the three germ layers, teratoma formation, chimera participation, germline transmission [see, e.g., Maherali and Hochedlinger, Cell Stem Cell, 3(6):595-605 (2008)], and tetraploid complementation.

[0127] Human iPSCs can be obtained using similar transduction methods, and the trio of transcription factors, OCT4, SOX2, and NANOG, have been established as a core set of transcription factors governing pluripotency (e.g., Budniatzky and Gepstein, Stem Cell Transl Med., 3(4):448-57 (2014); Barrett et al., Stem Cell Trans Med. 3:1-6, sctm.2014-0121 (2014); Focosi et al., Blood Cancer Journal, 4:e211 (2014); and references cited therein.) Generation of iPSCs can be achieved conventionally by using viral vectors to introduce nucleic acid sequences encoding stem cell-associated genes into adult somatic cells.

[0128] iPSCs can be generated or derived from terminally differentiated somatic cells, as well as from adult or somatic stem cells. That is, non-pluripotent progenitor cells can be reprogrammed to become pluripotent or multipotent. In such cases, it may not be necessary to include as many reprogramming factors as are required to reprogram terminally differentiated cells. Furthermore, reprogramming can be induced by non-viral introduction of reprogramming factors, for example, by introducing the protein itself, or by introducing a nucleic acid encoding the reprogramming factor, or by introducing a messenger RNA that, upon translation, provides the reprogramming factor (see, e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010)). Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-related genes, including, for example, Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. Reprogramming using the methods and compositions described herein may further include introducing one or more of Oct-3 / 4, a member of the Sox family, a member of the Klf family, and a member of the Myc family into somatic cells. The methods and compositions described herein may further include introducing one or more of Oct-4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As mentioned above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from the reprogrammed cells are to be used, for example, in human therapy, in one aspect, the reprogramming is not performed by a method that alters the genome, and thus, in such instances, reprogramming can be achieved without the use of, for example, viral or plasmid vectors.

[0129] The efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a starting cell population is measured by Shi et al., Cell-Stem Cell, 2:525-528 (2008); Huangfu et al. As shown by [End Page 110], Nature Biotechnology, 26(7):795-797 (2008); and Marson et al., Cell-Stem Cell, 3:132-135 (2008), the reprogramming efficiency can be enhanced by the addition of various agents, such as small molecules. Thus, agents or combinations of agents that enhance the efficiency or speed of induced pluripotent stem cell generation can be used in generating patient-specific or disease-specific iPSCs. Some non-limiting examples of agents that enhance reprogramming efficiency include, among others, soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase inhibitor), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA).

[0130] Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate and valproic acid (VPA) and other short-chain fatty acids), Scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, Apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g., 6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, and CHAP50. Other reprogramming enhancers include, for example, dominant-negative forms of HDACs (e.g., catalytically inactive forms), siRNA-based HDAC inhibitors, and antibodies that specifically bind to HDACs. Such inhibitors are available from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.

[0131] To confirm the induction of pluripotent stem cells for use in the methods described herein, isolated clones can be examined for the expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from a non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include immunological methods that detect the presence of encoded polypeptides, such as RT-PCR and Western blot or flow cytometry analysis. Detection can involve not only RT-PCR but also the detection of protein markers. Intracellular markers can best be identified through protein detection methods such as RT-PCR or immunocytochemistry, while cell surface markers are readily identified by, for example, immunocytochemistry.

[0132] The pluripotent stemness of isolated cells can be confirmed by testing the ability of iPSCs to differentiate into the cells of each of the three germ layers.For example, the pluripotency of isolated clones can be assessed using teratoma formation in nude mice.Cells can be introduced into nude mice, and histology and / or immunohistochemistry can be performed on tumors arising from cells.The growth of tumors that contain cells derived from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0133] Creation of DMD patient-specific iPSCs

[0134] One step of the ex vivo method of the present disclosure can involve generating a DMD patient-specific iPS cell, multiple DMD patient-specific iPS cells, or a DMD patient-specific iPS cell line. Many methods for generating patient-specific iPS cells have been established in the art, as described in Takahashi and Yamanaka, 2006; Takahashi, Tanabe et al., 2007. Additionally, differentiation of pluripotent cells into muscle lineages can be achieved by a technology developed by Anagenesis Biotechnologies, as described in International Patent Application Publication Nos. WO2013 / 030243 and WO2012 / 101114. For example, the generating step can include: a) isolating somatic cells, such as skin cells or fibroblasts, from a patient; and b) introducing a set of pluripotency-associated genes into the somatic cells to induce them to become pluripotent stem cells. The set of pluripotency-associated genes may be one or more of the genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

[0135] Genome editing

[0136] Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably in a precise or predetermined manner.The example of genome editing method described herein includes the method of using site-specific nucleases to cut deoxyribonucleic acid (DNA) at precise target positions in the genome, thereby creating single-stranded or double-stranded DNA breaks at specific positions in the genome.Such cutting has been recently described by Cox et al., Nature Medicine, Vol. 21 (No. 2) As reviewed in [Endogenous DNA Repair (2015)], pp. 121-31, repair can be and is regularly performed by natural endogenous cellular processes, such as homology-directed repair (HDR) and nonhomologous end joining (NHEJ). NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes accompanied by the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR utilizes a homologous or donor sequence as a template for inserting a defined DNA sequence into the breakpoint. The homologous sequence may be present within the endogenous genome, such as a sister chromatid. Alternatively, the donor may be an exogenous nucleic acid, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, or virus, that has a large region of homology with the locus to be cleaved by the nuclease but may also contain additional sequences or sequence changes, including deletions, that can be integrated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as "alternative NHEJ," which has a genetic outcome similar to NHEJ in that small deletions and insertions can occur at the break site. MMEJ can use a small number of base pairs of homologous sequences flanking the DNA break site to drive a more favorable repair outcome by joining the DNA ends, and recent reports have further elucidated the molecular mechanism of this process (e.g., Cho and Greenberg, Nature, 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online). doi:10.1038 / nsmb.2961 (2015); Mateos-Gomez et al., Nature 518:254-57 (2015); Ceccaldi et al., Nature 528:258-62 (2015). In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the DNA break site.

[0137] Each of these genome editing mechanisms can be used to create desired genome changes.The step in genome editing process can be to create one DNA break or two DNA breaks, as double-strand breaks or two single-strand breaks, in the target locus close to the intended mutation site.This can be achieved through the use of site-specific polypeptides, as described and exemplified herein.

[0138] Site-specific polypeptides such as DNA endonucleases can introduce double-strand or single-strand breaks into nucleic acids, for example, genomic DNA. The double-strand break can stimulate the cell's endogenous DNA repair pathways (e.g., homology-dependent repair, non-homologous end joining, alternative non-homologous end joining (A-NHEJ), or microhomology-mediated end joining). NHEJ can repair the cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) at the cleavage site within the target nucleic acid, which can disrupt or alter gene expression. HDR can occur when a homologous repair template or donor is available. The homologous donor template can contain sequences that can be homologous to sequences flanking the target nucleic acid cleavage site. Sister chromatids can be used by cells as repair templates. However, for genome editing purposes, repair templates can be provided as exogenous nucleic acids, such as plasmids, double-stranded oligonucleotides, single-stranded oligonucleotides, double-stranded oligonucleotides, or viral nucleic acids. Additional or modified nucleic acid sequences can also be introduced into the target locus, along with the exogenous donor template. Additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) can be introduced between the homologous flanking regions. MMEJ can produce genetic results similar to NHEJ in that small deletions and insertions can occur at the break site. MMEJ can use a small number of base pairs of homologous sequences flanking the break site to drive favorable end-joining DNA repair results. In some cases, it may be possible to predict likely repair outcomes based on analysis of potential microhomologies within the nuclease target region.

[0139] Therefore, in some cases, homologous recombination can be used to insert exogenous polynucleotide sequences into target nucleic acid cleavage sites.In this specification, exogenous polynucleotide sequences are referred to as donor polynucleotides (or donors or donor sequences or polynucleotide donor templates).Donor polynucleotides, parts of donor polynucleotides, copies of donor polynucleotides, or parts of copies of donor polynucleotides can be inserted into target nucleic acid cleavage sites.Donor polynucleotides can be exogenous polynucleotide sequences, i.e., sequences that do not naturally occur at target nucleic acid cleavage sites.

[0140] Modification of target DNA by NHEJ and / or HDR can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation. Deleting genomic DNA and integrating non-natural nucleic acids into genomic DNA are examples of genome editing.

[0141] CRISPR endonuclease system

[0142] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci encode products that function as a kind of immune system, helping to defend prokaryotes against foreign invaders, such as viruses and phages. There are three stages of CRISPR locus function: integration of new sequences into CRISPR loci, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acids. Five types of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

[0143] CRISPR loci contain several short repetitive sequences called "repeats." When expressed, repeats may form secondary structures (e.g., hairpins) and / or constitute unstructured single-stranded sequences. Repeats usually occur in clusters and often vary significantly between species. Repeats are regularly spaced by unique intervening sequences called "spacers," resulting in a repeat-spacer-repeat locus architecture. The spacers are identical to or highly homologous to known foreign invader sequences. The spacer-repeat units encode crisprRNAs (crRNAs), which are processed into the mature form of the spacer-repeat units. The crRNA contains a "seed" or spacer sequence (in its naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid) involved in targeting the target nucleic acid. The spacer sequence is located at the 5' or 3' end of the crRNA.

[0144] CRISPR locus also includes polynucleotide sequences encoding CRISPR-associated (Cas) genes. Cas genes encode endonucleases involved in the biosynthesis and interference stages of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and / or tertiary structures.

[0145] Type II CRISPR systems

[0146] In natural type II CRISPR systems, crRNA biogenesis requires tracrRNA (trans-activating CRISPR RNA). The tracrRNA can be modified by endogenous RNase III and then hybridize to crRNA repeats within the pre-crRNA array. Endogenous RNase III can be recruited to cleave the pre-crRNA. The cleaved crRNA can be subjected to exoribonuclease trimming (e.g., 5' trimming) to yield a mature crRNA form. The tracrRNA can further remain hybridized to the crRNA, and the tracrRNA and crRNA associate with a site-specific polypeptide (e.g., Cas9). The crRNA in the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA with the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid in type II CRISPR system is called protospacer adjacent motif (PAM). In nature, PAM is essential for facilitating the binding of site-specific polypeptide (such as Cas9) to target nucleic acid. Type II system (also called Nmeni or CASS4) can be further divided into type II-A (CASS4) and type II-B (CASS4a). Jinek et al., Science, vol. 337 (6096): pp. 816-821 (2012) demonstrates that CRISPR / Cas9 system is useful for RNA-programmable genome editing, and International Patent Application Publication No. WO2013 / 176772 presents many examples and applications of CRISPR / Cas endonuclease system for site-specific gene editing.

[0147] V-type CRISPR system

[0148] Type V CRISPR systems have several key differences from type II systems. For example, Cpf1 is a single RNA-guided endonuclease that lacks a tracrRNA, in contrast to type II systems. Indeed, Cpf1-associated CRISPR arrays can be processed into mature crRNAs without the requirement for further transactivation of the tracrRNA. Type V CRISPR arrays can be processed into short mature crRNAs, 42-44 nucleotides in length, with each mature crRNA beginning with a 19-nucleotide direct repeat followed by a 23-25 ​​nucleotide spacer sequence. In contrast, mature crRNAs in type II systems can begin with a 20-24 nucleotide spacer sequence followed by approximately 22 nucleotide direct repeats. Cpf1 also utilizes a T-rich protospacer adjacent motif, allowing the Cpf1-crRNA complex to efficiently cleave target DNA preceded by a short T-rich PAM, in contrast to type II systems, which have a G-rich PAM following the target DNA. Thus, type V systems cleave at a point distal to the PAM, whereas type II systems cleave at a point adjacent to the PAM. Additionally, in contrast to type II systems, Cpf1 cleaves DNA via a staggered double-strand break in the DNA with a 4- or 5-nucleotide 5' overhang. Type II systems cleave via a blunt-ended double-strand break. Like type II systems, Cpf1 contains a predicted RuvC-like endonuclease domain but lacks the second HNH endonuclease domain, in contrast to type II systems.

[0149] Cas gene / polypeptide and protospacer adjacent motifs

[0150] An exemplary CRISPR / Cas polypeptide is the Cas9 polypeptide in Figure 1 of Fonfara et al., Nucleic Acids Research, 42:2577-2590 (2014). The CRISPR / Cas system has been extensively rewritten since the discovery of Cas genes. Figure 5 in Fonfara, supra, shows the Cas9 gene family from various species. The PAM sequence of the polypeptide is presented.

[0151] Site-specific polypeptides

[0152] Site-specific polypeptide is the nuclease used in genome editing to cut DNA.Site-specific polypeptide can be administered to cells or patients as one or more polypeptides, or as one or more mRNAs encoding the polypeptide.

[0153] In the context of a CRISPR / Cas or CRISPR / Cpf1 system, the site-specific polypeptide is capable of binding to a guide RNA, which specifies a site within the target DNA to which the polypeptide is directed. In the CRISPR / Cas or CRISPR / Cpf1 systems disclosed herein, the site-specific polypeptide can be an endonuclease, such as a DNA endonuclease.

[0154] The site-directed polypeptide may comprise multiple nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains may be linked together via a linker. For example, the linker may comprise a flexible linker. The linker may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, or more amino acids in length.

[0155] The naturally occurring wild-type Cas9 enzyme contains two nuclease domains: an HNH nuclease domain and a RuvC domain. As used herein, "Cas9" refers to both naturally occurring Cas9 and recombinant Cas9. The Cas9 enzyme contemplated herein may contain an HNH or HNH-like nuclease domain, and / or a RuvC or RuvC-like nuclease domain.

[0156] The HNH or HNH-like domain contains an McrA-like fold. The HNH or HNH-like domain contains two antiparallel β-strands and an α-helix. The HNH or HNH-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The HNH or HNH-like domain can cleave one strand of the target nucleic acid (e.g., the complementary strand of the strand targeted by the crRNA).

[0157] The RuvC or RuvC-like domain contains an RNase H or RNase H-like fold. The RuvC / RNase H domain is involved in a diverse set of nucleic acid-based functions, including actions on both RNA and DNA. The RNase H domain contains five β strands surrounded by multiple α helices. The RuvC / RNase H or RuvC / RNase H-like domain contains a metal-binding site (e.g., a divalent cation-binding site). The RuvC / RNase H or RuvC / RNase H-like domain can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

[0158] The site-specific polypeptide can introduce a double-strand break or a single-strand break into a nucleic acid, for example, into genomic DNA. The double-strand break can stimulate the cell's endogenous DNA repair pathway (e.g., homology-dependent repair (HDR) or non-homologous end joining (NHEJ) or alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining (MMEJ)). NHEJ can repair the cut target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) at the cut site within the target nucleic acid, which can disrupt or alter gene expression. HDR can occur when a homologous repair template or donor is available. The homologous donor template can contain sequences homologous to the sequences flanking the target nucleic acid cut site. Sister chromatids can be used by the cell as repair templates. However, for genome editing purposes, repair templates can be provided as exogenous nucleic acids, such as plasmids, double-stranded oligonucleotides, single-stranded oligonucleotides, or viral nucleic acids. Additional or modified nucleic acid sequences can also be introduced into the target locus, along with the exogenous donor template. Additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) can be introduced between the homologous flanking regions. MMEJ can produce genetic results similar to NHEJ in that small deletions and insertions can occur at the break site. MMEJ can use a small number of base pairs of homologous sequences flanking the break site to drive favorable end-joining DNA repair results. In some cases, it may be possible to predict likely repair results based on the analysis of potential microhomologies within the nuclease target region.

[0159] Therefore, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. Herein, the exogenous polynucleotide sequence is referred to as a donor polynucleotide (or donor or donor sequence). A donor polynucleotide, a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a portion of a copy of a donor polynucleotide can be inserted into the target nucleic acid cleavage site. A donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

[0160] Modification of target DNA by NHEJ and / or HDR can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation. Deleting genomic DNA and integrating non-natural nucleic acids into genomic DNA are examples of genome editing.

[0161] The site-directed polypeptide can comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity to exemplary wild-type site-directed polypeptides [e.g., Cas9 from S. pyogenes; SEQ ID NO: 8 in US2014 / 0068797; or Sapranauskas et al., Nucleic Acids Res, 39(21):9275-9282 (2011)], and a variety of other site-directed polypeptides.

[0162] The site-directed polypeptide comprises at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. The site-directed polypeptide may comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids. The site-directed polypeptide may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the HNH nuclease domain of the site-directed polypeptide. A site-directed polypeptide can comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the HNH nuclease domain of the site-directed polypeptide. A site-directed polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the RuvC nuclease domain of the site-directed polypeptide. The site-directed polypeptide comprises up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids within the RuvC nuclease domain of the site-directed polypeptide.

[0163] The site-directed polypeptide may comprise a modified form of an exemplary wild-type site-directed polypeptide. The modified form of an exemplary wild-type site-directed polypeptide may comprise a mutation that reduces the nucleic acid cleavage activity of the site-directed polypeptide. The modified form of an exemplary wild-type site-directed polypeptide may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of an exemplary wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form of the site-directed polypeptide may not have substantial nucleic acid cleavage activity. When the site-directed polypeptide is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as "enzymatically inactive."

[0164] A modified form of a site-directed polypeptide can include a mutation such that it can induce a single-strand break (SSB) on a target nucleic acid (e.g., by cleaving only one of the sugar-phosphate backbones of a double-stranded target nucleic acid). The mutation can result in less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nucleic acid cleavage activity of one or more of the multiple nucleic acid cleavage domains of a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). The mutation can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave a complementary strand of the target nucleic acid but reducing their ability to cleave a non-complementary strand of the target nucleic acid. The mutation can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave a non-complementary strand of the target nucleic acid but reducing their ability to cleave a complementary strand of the target nucleic acid. For example, residues within an exemplary wild-type S. pyogenes Cas9 polypeptide, such as AsplO, His840, Asn854, and Asn856, are mutated to inactivate one or more of the multiple nucleic acid cleavage domains (e.g., nuclease domains). The residues to be mutated may correspond to residues AsplO, His840, Asn854, and Asn856 within an exemplary wild-type S. pyogenes Cas9 polypeptide (e.g., as determined by sequence and / or structural alignment). Non-limiting examples of mutations include D10A, H840A, N854A, or N856A. One skilled in the art will recognize that mutations other than alanine substitutions may be suitable.

[0165] The D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to create a site-directed polypeptide that substantially lacks DNA cleavage activity. The H840A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to create a site-directed polypeptide that substantially lacks DNA cleavage activity. The N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to create a site-directed polypeptide that substantially lacks DNA cleavage activity. The N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to create a site-directed polypeptide that substantially lacks DNA cleavage activity. A site-directed polypeptide that includes one substantially inactive nuclease domain is referred to as a "nickase."

[0166] RNA-guided endonucleases, such as Cas9 nickase mutants, can be used to increase the specificity of CRISPR-mediated genome editing.Wild-type Cas9 is typically guided by a single guide RNA, which is designed to hybridize with a specified sequence of about 20 nucleotides within a target sequence (such as an endogenous genomic locus).However, some mismatches between the guide RNA and the target locus can be tolerated, effectively reducing the required homology length within the target site to, for example, a minimum of 13nt of homology, thereby increasing the potential for CRISPR / Cas9 complexes to bind and cleave double-stranded nucleic acid (also known as off-target cleavage) elsewhere within the target genome.Since each Cas9 nickase mutant cleaves only one strand, to create a double-stranded break, a pair of nickases must bind in close proximity to each other on opposite strands of the target nucleic acid, thereby creating a pair of nicks, which is the equivalent of a double-stranded break. This requires that two separate guide RNAs (one for each nickase) must be adjacent and bind on opposite strands of the target nucleic acid. This requirement essentially doubles the minimum homology length required for a double-strand break to occur, thereby reducing the likelihood that a double-strand break event will occur elsewhere in the genome if the two guide RNA sites (if present) are unlikely to be close enough to each other to allow the formation of a double-strand break. As described in the art, nickases can also be used to promote HDR compared to NHEJ. HDR can be used to introduce selected changes into target sites in the genome through the use of specific donor sequences that effectively mediate the desired changes. Various CRISPR / Cas systems for use in gene editing are described, for example, in International Patent Application Publication No. WO2013 / 176772; and Nature Biotechnology, Vol. 32, pp. 347-355 (2014); and the references cited therein. These can be found in the references cited herein.

[0167] Contemplated mutations may include substitutions, additions, and deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to a natural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid to an amino acid mimetic (e.g., a phosphomimetic). The mutation may be a conservative mutation. For example, mutations may convert the mutated amino acid to an amino acid similar in size, shape, charge, polarity, conformation, and / or to a rotamer of the mutated amino acid (e.g., a cysteine / serine mutation, a lysine / asparagine mutation, a histidine / phenylalanine mutation). Mutations may cause a shift in the reading frame and / or the creation of a premature stop codon. Mutations may cause changes to regulatory regions of genes or loci that affect the expression of one or more genes.

[0168] Site-directed polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive site-directed polypeptides) may target nucleic acids. Site-directed polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonucleases) may target DNA. Site-directed polypeptides (e.g., mutant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonucleases) may target RNA.

[0169] The site-directed polypeptide can include one or more non-native sequences (eg, the site-directed polypeptide is a fusion protein).

[0170] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 derived from a bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain).

[0171] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 derived from a bacterium (e.g., S. pyogenes) and two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain).

[0172] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 derived from a bacterium (e.g., S. pyogenes) and two nucleic acid cleavage domains, wherein one or both of the nucleic acid cleavage domains comprises at least 50% amino acid identity to a nuclease domain derived from a Cas9 derived from a bacterium (e.g., S. pyogenes).

[0173] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 derived from a bacterium (e.g., S. pyogenes), two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or linker that links the site-directed polypeptide to the non-native sequence.

[0174] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to Cas9 from a bacterium (e.g., S. pyogenes), two nucleic acid cleavage domains (i.e., the HNH domain and the RuvC domain), wherein the site-directed polypeptide comprises a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

[0175] The site-directed polypeptide can comprise an amino acid sequence comprising at least 15% amino acid identity to a Cas9 derived from a bacterium (e.g., S. pyogenes) and two nucleic acid cleavage domains (i.e., an HNH domain and a RuvC domain), wherein one of the nuclease domains comprises a mutation of aspartic acid 10 and / or one of the nuclease domains comprises a mutation of histidine 840, which mutation reduces the cleavage activity of the nuclease domain by at least 50%.

[0176] The one or more site-specific polypeptides, e.g., DNA endonucleases, can include two nickases that together create one double-strand break at a specific locus in the genome, or four nickases that together create or cause two double-strand breaks at a specific locus in the genome. Alternatively, a single site-specific polypeptide, e.g., a DNA endonuclease, can create or cause one double-strand break at a specific locus in the genome.

[0177] Genome-targeting nucleic acids

[0178] The present disclosure provides a genome-targeting nucleic acid that can direct the activity of an associated polypeptide (e.g., a site-directed polypeptide) to a specific target sequence within a target nucleic acid. The genome-targeting nucleic acid can be RNA. Herein, the genome-targeting RNA is referred to as a "guide RNA" or "gRNA." The guide RNA can include at least a spacer sequence that hybridizes with the target nucleic acid sequence of interest and a CRISPR repeat sequence. In a type II system, the gRNA also includes a second RNA called a tracrRNA sequence. In a type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In a type V guide RNA (gRNA), the crRNA forms a duplex. In either system, the duplex can bind to the site-directed polypeptide such that the guide RNA and the site-directed polypeptide form a complex. The genome-targeting nucleic acid can provide target specificity to the complex through its association with the site-directed polypeptide. Thus, the genome-targeting nucleic acid can direct the activity of the site-specific polypeptide.

[0179] Exemplary guide RNAs include spacer sequences in the sequence listing shown along with the genomic location of their target sequence within or near the dystrophin gene and the associated site of cleavage by Cas9, where the genomic location is based on the GRCh38 / hg38 human genome assembly.

[0180] Each guide RNA can be designed to include a spacer sequence that is complementary to the target sequence of its genome in or near the dystrophin gene.For example, each spacer sequence in the sequence listing can be assembled into a single-stranded guide RNA (sgRNA) (for example, RNA chimera) or crRNA (with corresponding tracrRNA).See Jinek et al., Science, vol. 337, pp. 816-821 (2012); and Deltcheva et al., Nature, vol. 471, pp. 602-607 (2011).

[0181] The genome-targeting nucleic acid can be a dual-molecule guide RNA. The genome-targeting nucleic acid can be a single-molecule guide RNA.

[0182] A dual-molecule guide RNA may comprise two strands of RNA. The first strand comprises, from 5' to 3', an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0183] A single-molecule guide RNA (sgRNA) in a Type II system may comprise, from 5' to 3', an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute to further functionality (e.g., stability) of the guide RNA. The single-molecule guide linker may link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

[0184] The single-molecule guide RNA (sgRNA) within the V-type system can contain, in the 5' to 3' direction, a minimal CRISPR repeat sequence and a spacer sequence.

[0185] For illustrative purposes, guide RNAs or other smaller RNAs used in the CRISPR / Cas / Cpf1 system can be easily synthesized by chemical means, as exemplified below and described in the art. While chemical synthesis procedures are continually expanding, purification of such RNAs by procedures such as high-performance liquid chromatography (HPLC; avoiding the use of gels, such as PAGE) tends to become more difficult as polynucleotide lengths increase well beyond 100 or so nucleotides. One approach used to generate longer RNAs is to create two or more molecules that are ligated together. Much longer RNAs, such as RNAs encoding Cas9 or Cpf1 endonucleases, are more easily generated enzymatically. Various types of RNA modifications, such as those described in the art that enhance stability, reduce the likelihood or severity of innate immune responses, and / or enhance other attributes, can be introduced during or after chemical synthesis and / or enzymatic generation of RNA.

[0186] Spacer extension sequence

[0187] In some examples of genome-targeting nucleic acids, the spacer extension sequence can modify activity, provide stability, and / or provide a location for modifying the genome-targeting nucleic acid. The spacer extension sequence can modify on- or off-target activity or specificity. In some examples, the spacer extension sequence can be provided. The spacer extension sequence can have a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, or 7000 or more nucleotides. The spacer extension sequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000, or more nucleotides. The spacer extension sequence can be less than 10 nucleotides in length. The spacer extension sequence can be between 10 and 30 nucleotides in length. The spacer extension sequence can be between 30 and 70 nucleotides in length.

[0188] The spacer extension sequence may include another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). This moiety may decrease or increase the stability of the nucleic acid targeting nucleic acid. This moiety may be a transcription terminator segment (i.e., a transcription termination sequence). This moiety may function in eukaryotic cells. This moiety may function in prokaryotic cells. This moiety may function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include a 5' cap (e.g., a 7-methylguanylate cap (m7G)), a riboswitch sequence (e.g., that allows for modulation of stability and / or modulation of accessibility of proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a location within a cell (e.g., the nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates detection of fluorescence, a sequence that allows for detection of fluorescence, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.).

[0189] Spacer sequence

[0190] The spacer sequence hybridizes with a sequence in the target nucleic acid of interest. The spacer of the genome targeting nucleic acid can interact with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0191] In the CRISPR / Cas system herein, a spacer sequence can be designed to hybridize with the target nucleic acid located 5' to the PAM of the Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have a mismatch. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, S. pyogenes recognizes a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, where R contains A or G, and N is any nucleotide, and N is immediately 3' to the target nucleic acid sequence targeted by the spacer sequence.

[0192] The target nucleic acid sequence may comprise 20 nucleotides. The target nucleic acid may comprise fewer than 20 nucleotides. The target nucleic acid may comprise more than 20 nucleotides. The target nucleic acid may comprise at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. In some examples, the target nucleic acid may comprise up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target nucleic acid sequence may comprise the 20 bases immediately 5' to the first nucleotide of the PAM. For example, 5'-NNNNNNNNNNNNNNNNNNNN NRG In a sequence including -3' (SEQ ID NO: 1,410,473), the target nucleic acid may include a sequence corresponding to multiple Ns, where N is any nucleotide and the underlined NRG sequence is the PAM of S. pyogenes.

[0193] The spacer sequence that hybridizes with the target nucleic acid may have a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, or from about 1 nt to about 25 nt. The spacer sequence may be 0 nt to about 40 nt, about 10 nt to about 35 nt, about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 19 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt. In some examples, the spacer sequence may contain 20 nucleotides. The spacer sequence may contain 19 nucleotides.

[0194] In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some examples, the percent complementarity between the spacer sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some instances, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the 5'-most six contiguous nucleotides of the target sequence on the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid may be at least 60% over approximately 20 contiguous nucleotides. The lengths of the spacer sequence and the target nucleic acid may differ by 1 to 6 nucleotides, which can be considered one or more bulges.

[0195] Spacer sequences can also be designed or selected using computer programs, which can use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, %GC, genomic frequency (e.g., sequences that are identical or similar but vary in one or more spots as a result of mismatches, insertions, or deletions), methylation status, and the presence of SNPs.

[0196] Minimal CRISPR repeat sequence

[0197] A minimal CRISPR repeat sequence is a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

[0198] The minimal CRISPR repeat sequence may comprise nucleotides capable of hybridizing with the minimal tracrRNA sequence in cells. The minimal CRISPR repeat sequence and the minimal tracrRNA sequence may form a duplex, i.e., a base-paired, double-stranded structure. Together, the minimal CRISPR repeat sequence and the minimal tracrRNA sequence may bind to a site-specific polypeptide. At least a portion of the minimal CRISPR repeat sequence may hybridize with the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence may comprise at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity with the minimal tracrRNA sequence. At least a portion of the minimal CRISPR repeat sequence can comprise up to about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the minimal tracrRNA sequence.

[0199] The minimum CRISPR repeat sequence may be about 7 nucleotides to about 100 nucleotides in length. For example, the length of the minimum CRISPR repeat sequence is about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. In some examples, the minimum CRISPR repeat sequence is about 9 nucleotides in length. The minimum CRISPR repeat sequence may be about 12 nucleotides in length.

[0200] The minimal CRISPR repeat sequence can be at least about 60% identical to a reference minimal CRISPR repeat sequence (e.g., a wild-type crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8 consecutive nucleotides. For example, the minimal CRISPR repeat sequence is at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to the reference minimal CRISPR repeat sequence over a stretch of at least 6, 7, or 8 consecutive nucleotides.

[0201] Minimal tracrRNA sequence

[0202] A minimal tracrRNA sequence can be a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from S. pyogenes).

[0203] The minimal tracrRNA sequence may contain nucleotides that hybridize with the minimal CRISPR repeat sequence in cells. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence form a duplex, i.e., a base-paired, double-stranded structure. Together, the minimal tracrRNA sequence and the minimal CRISPR repeat bind to the site-specific polypeptide. At least a portion of the minimal tracrRNA sequence may hybridize with the minimal CRISPR repeat sequence. The minimal tracrRNA sequence may be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimal CRISPR repeat sequence.

[0204] The minimal tracrRNA sequence can have a length of about 7 nucleotides to about 100 nucleotides. For example, the minimal tracrRNA sequence can be about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt in length. The minimal tracrRNA sequence can be about 9 nucleotides in length. The minimal tracrRNA sequence can be about 12 ... It can consist of nt 23 to 48 of tracrRNA.

[0205] The minimal tracrRNA sequence can be at least about 60% identical to a reference minimal tracrRNA (e.g., a wild-type tracrRNA from S. pyogenes) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal tracrRNA sequence can be at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to the reference minimal tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0206] The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise a double helix. The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimal CRISPR RNA and the minimal tracrRNA may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0207] A duplex may contain mismatches (i.e., the two strands of the duplex are not 100% complementary). A duplex may contain at least about one, two, three, four, or five or more mismatches. A duplex may contain up to about one, two, three, four, or five or more mismatches. A duplex may contain no more than two mismatches.

[0208] bulge

[0209] In some cases, a "bulge" may exist within the duplex between the minimal CRISPR RNA and the minimal tracrRNA. A bulge is an unpaired region of nucleotides within the duplex. The bulge may contribute to the binding of the duplex to the site-specific polypeptide. The bulge may contain an unpaired 5'-XXXY-3' (wherein X is any purine and Y contains a nucleotide that can form a wobble pair with a nucleotide on the opposite strand) on one side of the duplex and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on the two sides of the duplex may differ.

[0210] In one example, the bulge may include an unpaired purine (e.g., adenine) on the minimal CRISPR repeat strand of the bulge. In some examples, the bulge may include an unpaired 5'-AAGY-3' (wherein Y includes a nucleotide that can form a wobble pair with a nucleotide on the minimal CRISPR repeat strand) on the minimal tracrRNA sequence strand of the bulge.

[0211] The bulge on the side of the duplex that is closest to the minimal CRISPR repeat may contain at least one, two, three, four, or five or more unpaired nucleotides.The bulge on the side of the duplex that is closest to the minimal CRISPR repeat may contain at most one, two, three, four, or five or more unpaired nucleotides.The bulge on the side of the duplex that is closest to the minimal CRISPR repeat may contain one unpaired nucleotide.

[0212] The bulge on the side of the duplex facing the minimal tracrRNA sequence may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. The bulge on the side of the duplex facing the minimal tracrRNA sequence may contain at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. The bulge on the second side of the duplex (e.g., on the side of the duplex facing the minimal tracrRNA sequence) may contain four unpaired nucleotides.

[0213] The bulge may include at least one wobble pairing. In some examples, the bulge may include at most one wobble pairing. In some examples, the bulge may include at least one purine nucleotide. The bulge may include at least three purine nucleotides. The bulge sequence may include at least five purine nucleotides. The bulge sequence may include at least one guanine nucleotide. The bulge sequence may include at least one adenine nucleotide.

[0214] hairpin

[0215] In various examples, one or more hairpins can be located 3' to the minimal tracrRNA within the 3' tracrRNA sequence.

[0216] The hairpin can begin at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides 3' of the last paired nucleotide in the duplex between the minimal CRISPR repeat and the minimal tracrRNA sequence. The hairpin can begin at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides 3' of the last paired nucleotide in the duplex between the minimal CRISPR repeat and the minimal tracrRNA sequence.

[0217] A hairpin can contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides. A hairpin can contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutive nucleotides.

[0218] The hairpin may contain a CC dinucleotide (ie, two consecutive cytosine nucleotides).

[0219] The hairpin may comprise duplex nucleotides (e.g., nucleotides hybridized together within the hairpin). For example, the hairpin may comprise a CC dinucleotide hybridized to a GG dinucleotide within the hairpin duplex of the 3' tracrRNA sequence.

[0220] One or more of the hairpins may interact with a region of the site-directed polypeptide that interacts with the guide RNA.

[0221] In some instances, there are two or more hairpins, and in other instances, there are three or more hairpins.

[0222] 3'tracrRNA sequence

[0223] The 3' tracrRNA sequence can include a sequence with at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA derived from S. pyogenes).

[0224] The 3' tracrRNA sequence can be about 6 nucleotides to about 100 nucleotides in length. For example, the 3' tracrRNA sequence can be about 6 nucleotides (nt) to about 50 nt, about 6 nt to about 40 nt, about 6 nt to about 30 nt, about 6 nt to about 25 nt, about 6 nt to about 20 nt, about 6 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. The 3' tracrRNA sequence can be about 14 nucleotides in length.

[0225] The 3' tracrRNA sequence can be at least about 60% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence derived from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the 3' tracrRNA sequence can be at least about 60% identical, 65% identical, 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 98% identical, 99% identical, or 100% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence derived from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0226] The 3' tracrRNA sequence may contain more than one duplex region (e.g., hairpin region, hybridizing region). The 3' tracrRNA sequence may contain two duplex regions.

[0227] The 3' tracrRNA sequence may comprise a stem-loop structure. The stem-loop structure within the 3' tracrRNA may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more nucleotides. The stem-loop structure within the 3' tracrRNA may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The stem-loop structure may comprise a functional moiety. For example, the stem-loop structure may comprise an aptamer, a ribozyme, a protein-interacting hairpin, a CRISPR array, an intron, or an exon. The stem-loop structure may comprise at least about 1, 2, 3, 4, or 5 or more functional moieties. The stem-loop structure may comprise at most about 1, 2, 3, 4, or 5 or more functional moieties.

[0228] The hairpin in the 3' tracrRNA sequence can include a P domain. In some instances, the P domain can include a double-stranded region in the hairpin.

[0229] tracrRNA extension sequence

[0230] Whether the tracrRNA is in the context of a single-molecule guide or a dual-molecule guide, it can result in a tracrRNA extension sequence. The tracrRNA extension sequence can be from about 1 nucleotide to about 400 nucleotides in length. The tracrRNA extension sequence can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or greater than 400 nucleotides in length. The tracrRNA extension sequence can be from about 20 to about 5000 or more nucleotides in length. The tracrRNA extension sequence can be greater than 1000 nucleotides in length. The tracrRNA extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or more nucleotides. The tracrRNA extension sequence may have a length of less than 1000 nucleotides. The tracrRNA extension sequence may comprise a length of less than 10 nucleotides. The tracrRNA extension sequence may be 10-30 nucleotides in length. The tracrRNA extension sequence may be 30-70 nucleotides in length.

[0231] The tracrRNA extension sequence may contain functional moieties (e.g., stability control sequences, ribozymes, endoribonuclease binding sequences). Functional moieties may include transcription terminator segments (i.e., transcription termination sequences). Functional moieties may have a total length of about 10 nucleotides (nt) to about 100 nucleotides, about 10 nt to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt. Functional moieties may function in eukaryotic cells. The functional moiety can function in a prokaryotic cell. The functional moiety can function in both eukaryotic and prokaryotic cells.

[0232] Non-limiting examples of suitable tracrRNA extension functional portions include a 3' polyadenylation tail, a riboswitch sequence (e.g., allowing for regulation of stability and / or accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates detection of fluorescence, a sequence that allows for detection of fluorescence, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, transcriptional repressor, DNA methyltransferase, DNA demethylase, histone acetyltransferase, histone deacetylase, etc.). The tracrRNA extension sequence may include a primer binding site or a molecular index (e.g., a barcode sequence). The tracrRNA extension sequence may include one or more affinity tags.

[0233] Single molecule guide linker sequence

[0234] The linker sequence of the single molecule guide nucleic acid can have a length of from about 3 nucleotides to about 100 nucleotides. Jinek et al., supra, for example, describe a simple four-nucleotide "tetraloop" (-GAAA-) was used (Science, Vol. 337 (No. 6096): pp. 816-821 (2012)). Exemplary linkers have a length of about 3 nucleotides (nt) to about 90 nt, about 3 nt to about 80 nt, about 3 nt to about 70 nt, about 3 nt to about 60 nt, about 3 nt to about 50 nt, about 3 nt to about 40 nt, about 3 nt to about 30 nt, about 3 nt to about 20 nt, or about 3 nt to about 10 nt. For example, the linker can have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. The linker of the single-molecule guide nucleic acid can be between 4 and 40 nucleotides. The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0235] The linker can comprise any of a variety of sequences, but in some instances, the linker does not comprise a sequence having extensive regions that are homologous to other portions of the guide RNA and that could cause intramolecular binding that could interfere with other functional regions of the guide. In the previous paper, a simple tetranucleotide sequence, -GAAA-, was used (Science, Vol. 337 (No. 6096): pp. 816-821 (2012)), but longer sequences may also be included. Many other arrangements may be used as well.

[0236] Linker sequence can comprise functional moiety.For example, linker sequence can comprise one or more features, including aptamer, ribozyme, hairpin that interacts with protein, protein binding site, CRISPR array, intron or exon.Linker sequence can comprise at least about 1, 2, 3, 4, or 5 or more functional moiety.In some examples, linker sequence can comprise at most about 1, 2, 3, 4, or 5 or more functional moiety.

[0237] A genome engineering strategy that corrects cells by deleting (excising), inserting, or replacing (deleting and inserting) one or more exons, or the splice acceptor or donor site of an aberrant intron.

[0238] The steps of the disclosed ex vivo methods involve using genome engineering to edit / correct the genome of DMD patient-specific iPS cells. Similarly, the steps of the disclosed in vivo methods involve using genome engineering to edit / correct the genome of muscle cells in DMD patients. Similarly, the steps of the disclosed cellular methods involve editing / correcting the dystrophin gene in human cells by genome engineering.

[0239] DMD patients exhibit a wide range of mutations within the dystrophin gene. Therefore, different patients will generally require different correction strategies. Any CRISPR endonuclease can be used in the methods of the present disclosure, and each CRISPR endonuclease has its own associated PAM, which may or may not be disease-specific. For example, gRNA spacer sequences for targeting the dystrophin gene with CRISPR / Cas9 endonuclease derived from S. pyogenes are identified in SEQ ID NOS: 1-467,030 and 1,410,430-1,410,472 of the Sequence Listing. gRNA spacer sequences for targeting the dystrophin gene with CRISPR / Cas9 endonuclease derived from S. aureus are identified in SEQ ID NOS: 467,031-528,196 of the Sequence Listing. gRNA spacer sequences for targeting the dystrophin gene with the CRISPR / Cas9 endonuclease derived from S. thermophilus are identified in SEQ ID NOs: 528,197 to 553,198 in the Sequence Listing. gRNA spacer sequences for targeting the dystrophin gene with the CRISPR / Cas9 endonuclease derived from T. denticola are identified in SEQ ID NOs: 553,199 to 563,911 in the Sequence Listing. gRNA spacer sequences for targeting the dystrophin gene with the CRISPR / Cas9 endonuclease derived from N. meningitides are identified in SEQ ID NOs: 563,912 to 627,854 and 1,410,400 to 1,410,402 in the Sequence Listing. The gRNA spacer sequences for targeting the dystrophin gene with CRISPR / Cpf1 endonucleases from Acidominoccoccus, Lachnospiraceae, and Franciscella Novicida are identified in the Sequence Listing as SEQ ID NOs: 627,855 to 1,410,399 and 1,410,403 to 1,410,429.

[0240] One genome engineering strategy involves exon deletion. Targeted deletion of specific exons may be an attractive strategy for treating a large subset of patients with a single therapeutic cocktail. By restoring the dystrophin reading frame, single-exon deletions are predicted to treat up to 13% of patients, whereas multi-exon deletions are predicted to treat up to 62% of patients. Multi-exon deletions may reach a larger number of patients, but the efficiency of larger deletions decreases significantly with increasing size. Therefore, preferred deletions may range in size from 400 to 350,000 base pairs (bp). For example, deletions can range in size from 400 to 1,000; 1,000 to 5,000; 5,000 to 10,000; 10,000 to 25,000; 25,000 to 50,000; 50,000 to 100,000; 100,000 to 200,000; or 200,000 to 350,000 base pairs.

[0241] As previously stated, the DMD gene contains 79 exons. Any one or more of the 79 exons or the splice acceptor or donor site of the aberrant intron can be deleted to restore the dystrophin reading frame. These methods provide gRNA pairs that can be used to delete exons 2, 8, 43, 44, 45, 46, 50, 51, 52, 53, 70, 45-53, or 45-55, as these are the regions predicted to reach the largest subset of patients (see Tables 1 and 2; the percentages given in Table 2 are averages reported in the literature).

[0242] Different regions of the DMD gene can be repaired by deletion and / or HDR. A specific combination of gRNAs that cut within the genomic region of interest can be used to correct mutations within the target exon. Coordinates are based on the GRch38 / hg38 genome assembly (Table 1). [Table 1] [Table 2]

[0243] The method provides a gRNA pair that deletes exon 2 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 2 and the other gRNA cutting at the 3' end of exon 2.

[0244] The method provides a gRNA pair that deletes exon 8 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 8 and the other gRNA cutting at the 3' end of exon 8.

[0245] The method provides a gRNA pair that deletes exon 43 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 43 and the other gRNA cutting at the 3' end of exon 43.

[0246] The method provides a gRNA pair that deletes exon 44 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 44 and the other gRNA cutting at the 3' end of exon 44.

[0247] The method provides a gRNA pair that deletes exon 45 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 45 and the other gRNA cutting at the 3' end of exon 45.

[0248] The method provides a gRNA pair that deletes exon 46 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 46 and the other gRNA cutting at the 3' end of exon 46.

[0249] The method provides a gRNA pair that deletes exon 50 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 50 and the other gRNA cutting at the 3' end of exon 50.

[0250] The method provides a gRNA pair that deletes exon 51 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 51 and the other gRNA cutting at the 3' end of exon 51.

[0251] The method provides a gRNA pair that deletes exon 52 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 52 and the other gRNA cutting at the 3' end of exon 52.

[0252] The method provides a gRNA pair that deletes exon 53 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 53 and the other gRNA cutting at the 3' end of exon 53.

[0253] The method provides a gRNA pair that deletes exon 70 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 70 and the other gRNA cutting at the 3' end of exon 70.

[0254] The method provides a gRNA pair that deletes exons 45 to 53 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 45 and the other gRNA cutting at the 3' end of exon 53.

[0255] The method provides a gRNA pair that deletes exons 45 to 55 by cleaving the gene twice, with one gRNA cleaving at the 5' end of exon 45 and the other gRNA cleaving at the 3' end of exon 55.

[0256] Another genome manipulation strategy involves inserting or replacing one or more exons or aberrant introns into splice acceptor or donor sites by homology-directed repair (HDR), also known as homologous recombination (HR). Homology-directed repair is one strategy for treating patients with premature stop codons due to small insertions / deletions or point mutations. Rather than causing large genomic deletions that convert the DMD phenotype to a BMD phenotype, this strategy restores the entire reading frame, completely reversing the disease state. This strategy requires a more customized approach based on the location of the premature stop for each patient. Most dystrophin exons are small (<300 bp). This is advantageous because HDR efficiency is inversely proportional to the size of the donor molecule. It is also expected that the donor template will be compatible with size-constrained adeno-associated virus (AAV) molecules, which have been shown to be an effective means of donor template delivery.

[0257] Homology-directed repair is a cellular mechanism for repairing double-strand breaks (DSBs). The most common form is homologous recombination. Additional pathways of HDR exist, including single-strand annealing and alternative HDR. Genome engineering tools allow researchers to manipulate the cellular homologous recombination pathway to create site-specific modifications to the genome. It has been discovered that cells can repair double-strand breaks using synthetic donor molecules supplied in trans. Thus, by introducing a double-strand break near a specific mutation and supplying an appropriate donor, targeted changes can be made in the genome. Specific breaks can be repaired in a manner similar to cells supplied with a homologous donor alone. 6This increases the HDR rate by more than 1,000-fold compared to the rate of 1 in 1. Because the rate of homology-guided repair (HDR) at a specific nucleotide is a function of the distance to the break site, it is important to select overlapping or nearest target sites. Gene editing offers an advantage over gene addition because in situ correction leaves the remainder of the genome undisturbed.

[0258] Donors provided for HDR editing vary significantly but can contain sequences with small or large flanking homology arms, intended to allow annealing to genomic DNA. The homology regions flanking the genetic changes to be introduced can be as small as 30 bp or smaller, or as large as multi-kilobase cassettes, which can contain promoters, cDNA, and other components. Both single-stranded and double-stranded oligonucleotide donors have been used. These oligonucleotides can range in size from less than 100 nt to more than 200 nt, although longer ssDNA can also be produced and used. Double-stranded donors, including PCR amplicons, plasmids, and minicircles, can be used. AAV vectors have generally been found to be highly effective means of delivering donor templates, although the packaging limit for individual donors is <5 kb. Active donor transcription increased HDR by 3-fold, indicating that including a promoter can enhance conversion. Conversely, methylation of donor CpGs reduces gene expression and HDR.

[0259] In addition to wild-type endonucleases such as Cas9, there are nickase mutants that can inactivate one or the other nuclease domain, resulting in cleavage of only one DNA strand. HDR can be induced by individual Cas nickases or by using a pair of nickases that flank the target region. The donor can be single-stranded, nicked, or dsDNA.

[0260] Donor DNA can be provided together with nuclease, or can be provided independently by various different methods, such as transfection, nanoparticle, microinjection or virus-mediated transduction.A range of tethering options have been proposed to increase the availability of donor for HDR.Examples include: donor is conjugated to nuclease, conjugated to the DNA binding protein that binds to the vicinity, or conjugated to the protein that is involved in the binding or repair at DNA end.

[0261] The choice of repair pathway can be guided by several culture conditions, such as those affecting the cell cycle, or by targeting DNA repair and related proteins: for example, key NHEJ molecules such as KU70, KU80, or DNA ligase IV can be inhibited to increase HDR.

[0262] In the absence of a donor, several non-homologous repair pathways can be used to join ends resulting from DNA breaks or from different breaks, which join DNA ends with little or no base pairing at the junction. In addition to canonical NHEJ, similar repair mechanisms exist, such as alt-NHEJ. When two breaks exist, the intervening segment can be deleted or inverted. NHEJ repair pathways can result in insertions, deletions, or mutations at the junction.

[0263] NHEJ was used to insert a 15 kb inducible gene expression cassette into a defined locus in a human cell line after nuclease cleavage (Maresca, M., Lin, V.G., Guo, N., and Yang, Y., Obligate ligation-gated recombination). (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining, Genome Res, vol. 23, pp. 539-546 (2013 year)).

[0264] In addition to genome editing by NHEJ or HDR, site-specific gene insertion has been carried out by using both NHEJ pathway and HR.Possibly, in certain situations, including intron / exon boundary, combination method may be applicable.NHEJ proves to be effective for ligation within intron, while error-free HDR may be better suited within coding region.

[0265] As previously stated, the DMD gene contains 79 exons. Any one or more of the 79 exons can be modified to correct the mutation and restore the dystrophin reading frame. Data indicates that the majority of premature stop codons in the dystrophin gene tend to be in exon 70, so some methods provide a gRNA or pair of gRNAs that can be used to facilitate the integration of new sequences derived from a polynucleotide donor template that insert or replace sequences within exon 70 (Tuffery-Giraud, S. et al., Hum Mutat 2009, 30(6):934-45) (Flanigan, KM et al., Hum Mutat 2009, 30(12):1657-66). To make the method applicable to the greatest number of patients, the method involves a donor template that can insert or replace the entire exon 70. Alternatively, the method provides a gRNA or pair of gRNAs that can be used to facilitate integration of new sequences derived from a polynucleotide donor template that insert or replace sequences within exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70. See Table 1.

[0266] After HDR, it is important to keep surrounding splicing signals intact to ensure proper processing of the pre-mRNA. Splicing donors and acceptors are generally within 100 base pairs of adjacent introns. Thus, in some instances, the method may result in all gRNAs cleaving approximately 0-3100 bp relative to the exon-intron junction.

[0267] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 2 and the other gRNA cutting at the 3' end of exon 2, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 2.

[0268] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 2.

[0269] Some example methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 8 and the other gRNA cutting at the 3' end of exon 8, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 8.

[0270] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 8.

[0271] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 43 and the other gRNA cutting at the 3' end of exon 43, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 43.

[0272] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 43.

[0273] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 44 and the other gRNA cutting at the 3' end of exon 44, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 44.

[0274] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from a polynucleotide donor template to replace sequence within exon 44.

[0275] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 45 and the other gRNA cutting at the 3' end of exon 45, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 45.

[0276] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 45.

[0277] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 46 and the other gRNA cutting at the 3' end of exon 46, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 46.

[0278] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 46.

[0279] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 50 and the other gRNA cutting at the 3' end of exon 50, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 50.

[0280] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 50.

[0281] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 51 and the other gRNA cutting at the 3' end of exon 51, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 51.

[0282] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 51.

[0283] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 52 and the other gRNA cutting at the 3' end of exon 52, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 52.

[0284] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 52.

[0285] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 53 and the other gRNA cutting at the 3' end of exon 53, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 53.

[0286] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 53.

[0287] Some methods provide a gRNA pair that cuts the gene twice to create a deletion, with one gRNA cutting at the 5' end of exon 70 and the other gRNA cutting at the 3' end of exon 70, facilitating the incorporation of new sequence derived from a polynucleotide donor template to replace the sequence within exon 70.

[0288] Alternatively, some methods provide a single gRNA according to the preceding paragraph that produces a single double-stranded break that facilitates insertion of new sequence derived from the polynucleotide donor template to replace the sequence within exon 70.

[0289] In addition to single-exon recombination by homology-guided repair, we also describe methods for performing partial cDNA knock-in of mutational hotspots found within the DMD gene. For example, treatment to repair exons 45-55 may treat up to 62% of patients. Rather than deleting or replacing exons 45-55 as described herein, another treatment option is to replace the entire exon 45-55 genomic region (including introns spanning >350,000 bp) with a cDNA containing only the coding region of exons 45-55, spanning approximately 1800 bp. Replacement can be performed using homology-guided repair methods. By excluding the intergenic region, the exon 45-55 cDNA (rather than the entire genomic region) can be more easily included with homology arms in any of the donor vectors described in the section of this application entitled "Nucleic Acids Encoding System Components." In this approach, two gRNAs and Cas9 or Cpf1 that remove the genomic region between exons 45 and 55 can be delivered along with a donor construct that replaces the deleted region with the desired cDNA knock-in.

[0290] Using the cDNA knock-in method, any series of exons can be replaced.

[0291] The cDNA knock-in sequence can be optimized to contain synthetic intron sequences. To ensure proper expression and processing of the DMD locus, synthetic introns smaller than the natural introns can be added between exons in the donor construct.

[0292] Exemplary modifications within the dystrophin gene include deletions, insertions, or recombinations within or proximal to the above-referenced dystrophin locus, such as within a region less than 3 kb, less than 2 kb, less than 1 kb, or less than 0.5 kb upstream or downstream of a specific exon. Given the relatively wide variation in mutations within the dystrophin gene, it will be appreciated that many of the above-referenced deletion, insertion, or recombination variations (including, without limitation, large deletions as well as small deletions) are expected to restore the dystrophin reading frame and restore the activity of the dystrophin protein.

[0293] Such variants can include deletions, insertions, or recombinations that are larger in the 5' and / or 3' direction than the specific exon in question, or smaller in either direction. Thus, "near" or "proximal" with respect to a deletion, insertion, or recombination of a specific exon means that the SSB or DSB locus associated with the boundary (also referred to herein as the endpoint) of the desired deletion, insertion, or replacement can be within a region of less than about 3 kb from the reference locus. The SSB or DSB locus can also be more proximal, within 2 kb, 1 kb, 0.5 kb, or 0.1 kb. In the case of small deletions, the desired endpoints can be at or "adjacent to" the reference locus, thereby meaning that the endpoints can be within 100 bp, 50 bp, 25 bp, or less than about 10 bp to 5 bp from the reference locus.

[0294] One advantage of duplicating or mimicking the product produced by exon skipping and / or restoring reading frame for patients with DMD is that it is safe and has already been known to be associated with the improvement of DMD.Other examples, including large or small deletions / insertions / recombinations, can be expected to bring the same benefit, as long as they restore the reading frame of dystrophin.Therefore, it can be expected that many variations of deletions, insertions and recombinations described and exemplified herein can be effective for improving DMD.

[0295] Selection of target sequence

[0296] Shifts in the location of the 5' and / or 3' boundaries relative to a particular reference locus can be used to facilitate or enhance particular applications of gene editing, which depend in part on the endonuclease system selected for editing, as further described and exemplified herein.

[0297] In a first non-limiting example of such target sequence selection, many endonuclease systems have rules or criteria that can guide the initial selection of potential target sites for cleavage, such as the requirement for a PAM sequence motif, particularly in the case of Type II or Type V CRISPR endonucleases, its location adjacent to the DNA cleavage site.

[0298] In another non-limiting example of target sequence selection or optimization, the frequency of "off-target" activity (i.e., the frequency of DSBs occurring at sites other than the selected target sequence) for a particular combination of target sequence and gene editing endonuclease can be evaluated relative to the frequency of on-target activity. In some cases, cells that have been properly edited at the desired locus may have a selective advantage over other cells. Illustrative, but non-limiting, examples of selective advantage include attributes such as enhanced replication rate, persistence, resistance to certain conditions, enhanced engraftment success or survival in vivo after introduction into a patient, and the acquisition of other attributes associated with the maintenance or increased number or viability of such cells. In other cases, cells that have been properly edited at the desired locus can be positively selected by one or more screening methods used to identify, sort, or otherwise select properly edited cells. Both selective advantage and directed selection methods may utilize phenotypes associated with correction. In some cases, cells can be edited twice or more times to create a second modification that creates a new phenotype that can be used to select or purify the intended cell population. Such a second modification can be created by adding a second gRNA for a selectable or screenable marker. In some cases, cells can be properly edited at the desired locus using a DNA fragment that contains a cDNA and also contains a selectable marker.

[0299] In certain cases, whether any selective advantage is applicable or any directed selection is applied, the selection of target sequence can also be guided by the consideration of off-target frequency, in order to enhance the effectiveness of application and / or reduce the potential of undesired changes at sites other than the desired target.As further described and illustrated herein and in the art, the occurrence of off-target activity can be influenced by several factors, including the similarity and difference between target site and various off-target sites, as well as the specific endonuclease used.Bioinformatics tools are available to help predict off-target activity, and these tools can also be used to identify the site with the highest probability of off-target activity, and then evaluate it under experimental conditions to assess the relative frequency of off-target to on-target activity, thereby enabling the selection of sequences with higher relative on-target activity.This paper provides an example of such technique, but other examples are known in the art.

[0300] Another aspect of target sequence selection relates to homologous recombination events. Sequences that share homologous regions can be used as a focus for homologous recombination events, resulting in the deletion of intervening sequences. Such recombination events occur during the normal course of replication of chromosomes and other DNA sequences, and occur periodically during the normal cell replication cycle, but also at other times when DNA sequences are synthesized, such as during the repair of double-strand breaks (DSBs), which can be enhanced by the occurrence of various events (such as UV light and other inducers of DNA breaks) or the presence of certain agents (such as various chemical inducers). Many such inducers cause DSBs indiscriminately within the genome, and DSBs can be induced and repaired regularly in normal cells. During repair, the original sequence can be reconstructed with complete fidelity, but in some cases, small insertions or deletions (called "indels") are introduced at the DSB site.

[0301] DSB can also be specifically induced at a particular location, as in the case of the endonuclease system described herein, and can be used to cause directed or preferential gene modification events at selected chromosomal locations.The tendency of homologous sequences to undergo recombination in DNA repair (and replication) can be utilized in some situations, and is the basis for one application of gene editing systems, such as CRISPR, which uses homology-guided repair to insert the sequence of interest provided through the use of a "donor" polynucleotide into a desired chromosomal location.

[0302] Small regions of "microhomology," which may be as few as 10 base pairs or less, can also be used to create the desired deletion between specific sequences. For example, a single DSB can be introduced at a site that exhibits microhomology with a neighboring sequence. During the normal course of repair of such DSBs, a frequent result is the deletion of the intervening sequence as a result of recombination facilitated by the DSB and concurrent cellular repair processes.

[0303] However, in some situations, selecting a target sequence within the region of homology may also result in much larger deletions, including gene fusions (if the deletion is within a coding region), which may or may not be desirable given the particular circumstances.

[0304] The examples presented herein further illustrate the selection of diverse target regions for creating DSBs designed to induce disruptions, deletions, or recombinations that result in restoration of the dystrophin reading frame, as well as the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events.

[0305] Nucleic acid modification

[0306] In some cases, the polynucleotide introduced into the cell may contain one or more modifications that may be used individually or in combination, for example, to enhance activity, stability, or specificity, alter delivery, reduce the innate immune response in the host cell, or other enhancements further described herein and known in the art.

[0307] In certain instances, modified polynucleotides may be used in a CRISPR / Cas9 / Cpf1 system, where the guide RNA (single-molecule guide or dual-molecule guide) and / or the DNA or RNA encoding the Cas or Cpf1 endonuclease introduced into a cell may be modified as described and exemplified below. Such modified polynucleotides may be used in a CRISPR / Cas9 / Cpf1 system to edit any one or more genomic loci.

[0308] Using the CRISPR / Cas9 / Cpf1 system as a non-limiting example of such use, guide RNA modification can be used to enhance the formation or stability of a CRISPR / Cas9 / Cpf1 genome editing complex, which may be a single-molecule guide or a double-molecule guide RNA and a Cas or Cpf1 endonuclease. Guide RNA modification can also or alternatively be used to enhance the initiation, stability, or kinetics of the interaction between the genome editing complex and a target sequence in the genome, which can be used, for example, to enhance on-target activity. Guide RNA modification can also or alternatively be used to enhance specificity, for example, the relative rate of genome editing at on-target sites compared to the effect at other (off-target) sites.

[0309] Modifications can also, or alternatively, be used to increase the stability of the guide RNA, for example, by increasing its resistance to degradation by ribonucleases (RNases) present in the cell, thereby extending its half-life within the cell. Modifications that extend the half-life of the guide RNA can be particularly useful in embodiments in which a Cas or Cpfl endonuclease is introduced into a cell to be edited via RNA that needs to be translated to generate the endonuclease. This is because the guide RNA half-life extension, which is introduced simultaneously with the RNA encoding the endonuclease, can be used to extend the time that the guide RNA and the encoded Cas or Cpfl endonuclease coexist within the cell.

[0310] Modifications can also, or alternatively, be used to reduce the likelihood or extent to which RNA introduced into a cell will induce an innate immune response. In the context of RNA interference (RNAi), including small interfering RNA (siRNA), such responses, which are well characterized and described below and in the art, tend to be associated with a shortened half-life of the RNA and / or the induction of cytokines or other factors associated with the immune response.

[0311] One or more modifications can also be made to the RNA encoding the endonuclease introduced into a cell, including, without limitation, modifications that enhance the stability of the RNA (such as by increasing its resistance to degradation by RNases present in the cell), modifications that enhance translation of the resulting product (i.e., the endonuclease), and / or modifications that reduce the likelihood or extent to which the RNA introduced into the cell will elicit a natural immune response.

[0312] Combinations of the above and other modifications can also be used. In the case of CRISPR / Cas9 / Cpfl, for example, one or more modifications can be made to the guide RNA (including the guide RNAs exemplified above) and / or one or more modifications can be made to the RNA encoding the Cas endonuclease (including the RNA encoding the Cas endonuclease exemplified above).

[0313] For illustrative purposes, guide RNAs or other small RNAs used in the CRISPR / Cas9 / Cpf1 system can be easily synthesized by chemical means, and as exemplified below and described in the art, some modifications can be easily incorporated. While chemical synthesis procedures are continuously expanding, the purification of such RNAs by procedures such as high performance liquid chromatography (HPLC; avoiding the use of gels, such as PAGE) tends to become more difficult as polynucleotide lengths increase well beyond about 100 nucleotides. One approach that can be used to generate longer chemically modified RNAs is to create two or more molecules that are ligated together. Much longer RNAs, such as RNAs encoding Cas9 endonuclease, are more easily generated enzymatically. Although a small number of modifications are available for use in enzymatically generated RNA, there are still modifications, and new types of modifications are being regularly developed, that can be used, for example, to enhance stability, reduce the likelihood or extent of an innate immune response, and / or enhance other attributes, as described below and further in the art.

[0314] For illustrative purposes, various types of modifications, particularly those frequently used with chemically synthesized small RNAs, can include one or more nucleotides modified at the 2' position of the sugar, in some embodiments, nucleotides modified with 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro. In some embodiments, RNA modifications can include 2'-fluoro, 2'-amino, or 2'-O-methyl modifications of the ribose of pyrimidines, abasic residues, or the reverse base at the 3' end of the RNA. Such modifications can be incorporated into oligonucleotides in a defined manner, and these oligonucleotides have been shown to have a higher Tm (i.e., a higher binding affinity to the target) than 2'-deoxyoligonucleotides for a given target.

[0315] Some nucleotide and nucleoside modifications have been shown to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligonucleotides, and these modified oligonucleotides remain intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified backbones, e.g., phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages. Some oligonucleotides include oligonucleotides with phosphorothioate backbones, and heteroatom backbones, particularly CH2-NH-O-CH2, CH, ~N(CH3)~O~CH2 (known as the methylene (methylimino) or MMI backbone), CH2--O--N(CH3)-CH2 backbone, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 backbones (where the natural phosphodiester backbone is represented as OPO-CH); amide backbones (see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); ); oligonucleotides with a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of an oligonucleotide is replaced with a polyamide backbone and the nucleotides are linked directly or indirectly to aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science, 1991, 254, 1497). Phosphorus-containing linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates, including 3' alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, including those with normal 3'-5' linkages, 2'-5' linked analogs thereof, and phosphorus-containing linkages with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Examples of suitable hydroxybenzoates include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5, (See Nos. 405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050).

[0316] Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14):4503-4510 (2002); Genesis, 30, 3 (2001); Heasman, Dev. Biol., 243:209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97:9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued July 23, 1991.

[0317] For cyclohexenyl nucleic acid oligonucleotide mimetics, see Wang et al., J. Am. Chem. Soc., 122:8595-8602 (2000).

[0318] Modified oligonucleotide backbones that do not contain a phosphorus atom have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatoms or heterocycles. These include backbones with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with a mixture of N, O, S, and CH component moieties (see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,214,134; each of which is incorporated herein by reference). No. 6,141; No. 5,235,033; No. 5,264,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; No. 5, No. 466,677; No. 5,470,967; No. 5,489,677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5 ,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439).

[0319] One or more substituted sugar moieties, such as one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCHOCH3, OCHO(CH2)nCH3, O(CH2)nNH2, or O(CH2)nCH3, where n is 1 to about 10; C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalating agent; group for improving the pharmacokinetic properties of the oligonucleotide; or group for improving the pharmacodynamic properties of the oligonucleotide; and other substituents with similar properties. In some embodiments, modifications include the 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl)) modification (Martin et al., HeIv. Chim. Acta, 1995, 78:486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of 5'-terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls, in place of pentofuranosyl groups.

[0320] In some cases, both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic, which has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide can be replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases can be retained and linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings on PNA compounds can be found in Nielsen et al., Science, 254:1497-1500 (1991).

[0321] Guide RNAs may also, additionally or alternatively, include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only infrequently or transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'deoxycytosine, and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazoylalkyl)adenine, 2-(aminoalklyamino)adenine or other hetero-substituted alkyladenines, 2-thiouracil, 2-methyl- ... Examples of base substitutions include uracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine (Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, pp. 75-77 (1980); Gebeyehu et al., Nucl. Acids Res., vol. 15:4513 (1997)). "Universal" bases known in the art, such as inosine, can also be incorporated. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, ST, and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions.

[0322] Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine, and 6-azothymine, 5-uracil (sh-uracil), uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl adenine and guanine and other 8-substituted adenines and guanines, 5-halouracil and cytosine, particularly 5-bromouracil and cytosine, 5-trifluoromethyluracil and cytosine and other 5-substituted uracils and cytosines, 7-methylquanine and adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, as well as other synthetic and naturally occurring nucleobases.

[0323] Further, the nucleobases may be any of the nucleobases disclosed in U.S. Pat. No. 3,687,808; the nucleobases disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed., John Wiley & Sons, 1990; the nucleobases disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, Vol. 30, p. 613; and and Sanghvi, YS, Chapter 15, "Antisense Research and Applications," pp. 289-302, Crooke, ST and Lebleu, B. (eds.), CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST). and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and more particularly when combined with 2'-O-methoxyethyl sugar modifications, are embodiments of base substitutions. Modified nucleobases are described in U.S. Patent Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; Nos. 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and U.S. Patent Application Publication No. 2003 / 0158403.

[0324] Thus, the term "modified" refers to an unnatural sugar, phosphate, or base that has been incorporated into the guide RNA, the endonuclease, or both the guide RNA and the endonuclease. Not all positions within a given oligonucleotide need be uniformly modified; in fact, more than one of the aforementioned modifications may be incorporated into a single oligonucleotide, or even into a single nucleoside within an oligonucleotide.

[0325] The guide RNA and / or mRNA (or DNA) encoding the endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties can be lipid moieties, such as cholesterol moieties [Letsinger et al., Proc. Natl. Acad., Sci. USA 86:6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let. 4:1053-1060 (1994)]; thioethers, e.g., hexyl-S-tritylthiol [Manoharan et al., Ann. NY Acad. Sci. 660:306-309 (1992); and Manoharan et al., Bioorg. Med. Chem. Let. 3:2765-2770 (1993)]; thiocholesterol [Oberhauser et al., Nucl. Acids Res. 20:533-538 (1992)]; aliphatic chains , for example, dodecanediol or undecyl residues [Kabanov et al., FEBS Lett., 259:327-330 (1990); and Svinarchuk et al., Biochimie, 75:49 54 (1993)]; phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36:3651-365 4 (1995); and Shea et al., Nucl. Acids Res., 18:3777-3783 (1990)]; polyamine or polyethylene glycol chains [Mancharan et al., Nucleosides & Nucleotides, 14:969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]. )]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264:229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937 (1996)]. Also, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022 ; Same No. 5,254,469; Same No. 5,258,506; Same No. 5,262,536; Same No. 5,272,250; Same No. 5,292,873; Same No. 5,317,098; Same No. 5,371,241; Same No. 5,391,723 See also Nos. 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941.

[0326] Sugars and other moieties can be used to target proteins and nucleotide-containing complexes, such as cationic polysomes and liposomes, to specific sites.For example, hepatocyte-directed import can be mediated by asialoglycoprotein receptor (ASGPR) (see, for example, Hu et al., Protein Pept Lett., vol. 21 (10): 1025-30 (2014)).Other systems known in the art and constantly being developed can be used to target the biomolecules and / or their complexes used in this case to specific target cells of interest.

[0327] These targeting moieties or conjugates may contain conjugate groups covalently attached to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the present invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In the context of the present disclosure, groups that enhance pharmacodynamic properties include groups that improve uptake, groups that enhance resistance to degradation, and / or groups that enhance sequence-specific hybridization with target nucleic acids. In the context of the present invention, groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism, or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed October 23, 1992, and U.S. Patent No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine chains or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.See, for example, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414 ,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,2 See Nos. 03; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941.

[0328] Longer polynucleotides, which are less amenable to chemical synthesis and are typically produced by enzymatic synthesis, can also be modified by a variety of means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other moieties at the 5' or 3' end of the molecule, and other modifications. By way of example, an mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied, for example, to increase its translation or stability (such as by increasing its resistance to cellular degradation), or to reduce the tendency of the RNA to elicit an innate immune response, which is often observed in cells after the introduction of exogenous RNA, particularly a long RNA such as an RNA encoding Cas9.

[0329] Many such modifications have been described in the art, including the use of poly-A tails, 5' cap analogs (e.g., ARCA (Anti-Reverse Cap Analog) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5'-triphosphate (5-methyl-CTP) or N6-methyl-ATP), or treatment with phosphatases to remove the 5'-terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are regularly developed.

[0330] There are numerous commercial sources of modified RNAs, including, for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon, and many others. As described by TriLink, for example, 5-methyl-CTP can be used to confer desirable characteristics such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with in vitro transcribed RNA. Kormann et al. and Warren et al., referenced below, As exemplified in publications such as

[10] , 5-methylcytidine-5'-triphosphate (5-methyl-CTP), N6-methyl-ATP, and pseudo-UTP and 2-thio-UTP have also been shown to enhance translation while reducing innate immune stimulation in culture and in vivo.

[0331] It has been shown that improved therapeutic efficacy can be achieved using chemically modified mRNA delivered in vivo (see, e.g., Kormann et al., Nature Biotechnology, vol. 29, pp. 154-157 (2011)). Such modifications can be used, for example, to increase the stability of RNA molecules and / or reduce their immunogenicity. Chemical modifications such as pseudo-U, N6-methyl-A, 2-thio-U, and 5-methyl-C have been found to result in a significant reduction in toll-like receptor (TLR)-mediated mRNA recognition in mice, by reducing the activation of the innate immune system (see, e.g., Kormann et al., supra).

[0332] It has also been shown that repeated administration of synthetic messenger RNA incorporating modifications designed to bypass the innate antiviral response can reprogram differentiated human cells to pluripotency. See, e.g., Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs, acting as primary reprogramming proteins, can be an efficient means of reprogramming multiple human cell types. These cells are termed induced pluripotent stem cells (iPSCs), and it has been discovered that enzymatically synthesized RNA incorporating 5-methyl-CTP, pseudo-UTP, and ARCA (Anti-Reverse Cap Analog) can be used to effectively evade the cellular antiviral response (see, e.g., Warren et al., supra).

[0333] Other modifications of polynucleotides described in the art include, for example, the use of a polyA tail, the addition of a 5' cap analog (such as m7G(5')ppp(5')G (mCAP)), modification of the 5' or 3' untranslated region UTR, or treatment with phosphatases to remove the 5' terminal phosphate (and new methods are regularly being developed).

[0334] Some compositions and techniques that can be applied to produce modified RNA for use herein have been developed in the context of RNA interference (RNAi) modification, including small interfering RNA (siRNA).The effect of siRNA on gene silencing via mRNA interference is generally transient, which may require repeated administration, so in vivo presents a particular challenge.In addition, siRNA is double-stranded RNA (dsRNA), and mammalian cells have evolved immune responses to detect and neutralize dsRNA, which is often the by-product of virus infection. Thus, there are mammalian enzymes, such as PKR (dsRNA-responsive kinase), that can mediate cellular responses to dsRNA, and potentially retinoic acid-inducible gene I (RIG-I), as well as Toll-like receptors (such as TLR3, TLR7, and TLR8) that can elicit cytokine induction in response to such molecules (e.g., Angart et al., Pharmaceuticals (Basel), 6(4):440-468 (2013); Kanasty et al., Molecular Therapy, 20(3):513-524 (2012); Burnett et al., Biotechnol J., 6(9):1130-46 (2011); Judge and MacLachlan Reviewed by , Hum Gene Ther, Vol. 19(No. 2): pp. 111-24 (2008); (See also the references cited therein).

[0335] A wide variety of modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that may be useful in connection with the introduction of polynucleotides into human cells as described herein (see, e.g., Whitehead KA et al., Annual Review of Chemical and Biomolecular Engineering, Vol. 2:7). 7-96 (2011); Gaglione and Messere, Mini Rev Med Chem, vol. 10 ( 7): pp. 578-95 (2010); Chernolovskaya et al., Curr Opin Mol Ther., vol. 12(2): pp. 158-67 (2010); Deleavy et al., Curr Protoc Nucleic Acids Chem, Chapter 16: Part 16.3 (2009); Behlke, Oligonucleotides, 18 Vol. (4): pp. 305-19 (2008); Fucini et al., Nucleic Acid Ther, Vol. 22 (No. 3): pp. 205-210 (2012); Bremsen et al., Front Genet, Vol. 3: 154 (See the review by Page (2012)).

[0336] As mentioned above, there are several commercial sources of modified RNAs, many of which specialize in modifications designed to improve siRNA efficacy. Various approaches are offered based on the diverse findings reported in the literature. For example, Dharmacon, as described in Kole, Nature Reviews Drug Discovery, 11:125-140 (2012),

[10] mentions that replacement of non-bridging oxygens with sulfur (phosphorothioate, PS) has been used to improve the nuclease resistance of siRNAs, as reported by Soutschek et al., Nature, 43. It has been reported that modification of the 2' position of ribose improves the nuclease resistance of the internucleotide phosphate bond while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. 2:173-178 (2004), the combination of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-methyl, 2'-fluoro, 2'-hydro) has been associated with highly stable siRNAs for in vivo applications, and Volkov, Oligonucleotides, 19:191-202 (2009), 2'-O-methyl modifications have been reported to be effective in improving stability. In the context of reducing the induction of innate immune responses, modifying specific sequences with 2'-O-methyl, 2'-fluoro, or 2'-hydro has been reported to reduce TLR7 / TLR8 interactions while generally preserving silencing activity (see, e.g., Judge et al., Mol. Ther., 13:494-505 (2006); and Cekaite et al., J. Mol. Biol., 365:90-108 (2007)). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine, have also been shown to minimize immune effects mediated by TLR3, TLR7, and TLR8 (see, e.g., Kariko, K. et al., Immunity, 23:165-175 (2005)). stomach).

[0337] Additionally, several conjugates known in the art and commercially available, including, for example, cholesterol, tocopherol, and folate, lipids, peptides, polymers, linkers, and aptamers, which may enhance their delivery and / or cellular uptake, can be applied to polynucleotides, such as RNA, for use herein (see, e.g., review by Winkler, Ther. Deliv., 4:791-809 (2013) and references cited therein).

[0338] Codon optimization

[0339] Polynucleotides encoding site-directed polypeptides can be codon-optimized for expression in cells containing the desired target DNA, according to standard methods in the art. For example, if the intended target nucleic acid is in a human cell, a codon-optimized human polynucleotide encoding Cas9 is contemplated for use to produce the Cas9 polypeptide.

[0340] Complex of genome-targeting nucleic acid and site-specific polypeptide

[0341] The genome-targeting nucleic acid interacts with a site-specific polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), thereby forming a complex. The genome-targeting nucleic acid guides the site-specific polypeptide to the target nucleic acid.

[0342] RNP

[0343] Each of the site-specific polypeptide and genome-targeting nucleic acid can be administered separately to cells or patients.On the other hand, the site-specific polypeptide can be pre-complexed with one or more guide RNAs or one or more crRNAs in combination with tracrRNA.The pre-complexed material can then be administered to cells or patients.This pre-complexed material is known as ribonucleoprotein particle (RNP).

[0344] Nucleic acids encoding components of the system

[0345] The present disclosure provides nucleic acids comprising nucleotide sequences encoding genome-targeting nucleic acids of the present disclosure, site-directed polypeptides of the present disclosure, and / or any nucleic acid or proteinaceous molecule necessary to carry out aspects of the methods of the present disclosure.

[0346] A nucleic acid encoding a genome-targeting nucleic acid of the present disclosure, a site-specific polypeptide of the present disclosure, and / or any nucleic acid or proteinaceous molecule necessary to carry out an embodiment of a method of the present disclosure may comprise a vector (e.g., a recombinant expression vector).

[0347] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional nucleic acid segments may be ligated. Another type of vector is a viral vector, into which additional nucleic acid segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0348] In some cases, vectors may be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or more simply, "expression vectors," although they serve equivalent functions.

[0349] The term "operably linked" means that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and can be found, for example, in Goeddel, Gene Expression Technology, Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA (1990) Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). One skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of target cell, the desired expression level, and the like.

[0350] Contemplated expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Additional vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3, which are described in Figures 1A-1C. Other vectors can be used as long as they are compatible with the host cell.

[0351] In some examples, vectors can comprise one or more transcription and / or translation control elements.Depending on the host / vector system used, any of several suitable transcription and translation control elements can be used in expression vectors, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc.The vector can be a self-inactivating vector, which inactivates viral sequences or components of CRISPR mechanism or other elements.

[0352] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include promoters derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) derived from retroviruses, the human elongation factor 1 promoter (EF1), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein I.

[0353] For expressing small RNAs, including guide RNAs used in conjunction with Cas endonucleases, various promoters may be advantageous, such as RNA polymerase III promoters, including, for example, U6 and H1. Descriptions of and parameters for enhancing the use of such promoters are known in the art, and additional information and techniques are regularly described (see, e.g., Ma, H. et al., Molecular Therapy - Nucleic Acids, 3, e161 (2014), doi:10.1038 / mtna.2014.12).

[0354] The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also contain a sequence suitable for amplifying expression. The expression vector may also contain a nucleotide sequence encoding a non-natural tag (e.g., a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.) that is fused to a site-specific polypeptide, thus resulting in a fusion protein.

[0355] The promoter may be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter may be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some cases, the promoter may be a spatially and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type specific promoter, etc.).

[0356] The genome-targeting nucleic acid and / or nucleic acid encoding the site-specific polypeptide of the present disclosure can be packaged in or on the surface of a delivery vehicle for delivery to cells. Possible delivery vehicles include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. Various targeting moieties can be used to enhance the preferential interaction of such vehicles with the desired cell type or location.

[0357] The complexes, polypeptides, and nucleic acids of the present disclosure can be introduced into cells by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0358] delivery

[0359] The guide RNA polynucleotide (RNA or DNA) and / or endonuclease polynucleotide (RNA or DNA) can be delivered by a virus or by a non-viral delivery vehicle known in the art. Alternatively, the endonuclease polypeptide can be delivered by a non-viral delivery vehicle known in the art, such as electroporation or lipid nanoparticles. In yet another alternative embodiment, the DNA endonuclease can be delivered as one or more polypeptides alone, or pre-complexed with one or more guide RNAs, or one or more crRNAs, in combination with tracrRNA.

[0360] Polynucleotide can be delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, vol. 18: 1127-1133 (2011) (which focuses on the non-viral delivery vehicles for siRNA, which are also useful for the delivery of other polynucleotides).

[0361] Polynucleotides such as guide RNAs, sgRNAs, and mRNAs encoding endonucleases can be delivered to cells or patients by lipid nanoparticles (LNPs).

[0362] LNP refers to any particle with a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, nanoparticles can range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm.

[0363] LNPs can be made from cationic lipids, anionic lipids, or neutral lipids. Neutral lipids such as the fusogenic phospholipid DOPE or membrane component cholesterol can be incorporated into LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. The limitations of cationic lipids include low efficacy due to low stability and rapid clearance, and the occurrence of inflammatory or anti-inflammatory responses.

[0364] LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0365] Any lipid or lipid combination known in the art can be used to prepare LNPs. Examples of lipids used to prepare LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0366] Lipids can be combined in any number of molar ratios to make LNPs. In addition, polynucleotides can be combined with lipids in a wide range of molar ratios to make LNPs.

[0367] As stated above, each of the site-specific polypeptide and genome-targeting nucleic acid can be administered to cells or patients separately.On the other hand, the site-specific polypeptide can be pre-complexed with one or more guide RNAs or one or more crRNAs in combination with tracrRNA.The pre-complexed material can then be administered to cells or patients.This pre-complexed material is known as ribonucleoprotein particle (RNP).

[0368] RNA can form specific interactions with either RNA or DNA. This property is utilized in many biological processes, but it also poses the risk of promiscuous interactions in the nucleic acid-rich intracellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs), in which RNA is pre-complexed with endonucleases. Another benefit of RNPs is the protection of RNA from degradation.

[0369] The endonuclease in RNP can be modified or unmodified.Similarly, gRNA, crRNA, tracrRNA or sgRNA can be modified or unmodified.Many modifications are known in the art and can be used.

[0370] The endonuclease and sgRNA can be combined in a 1:1 molar ratio. Alternatively, the endonuclease, crRNA, and tracrRNA can generally be combined in a 1:1:1 molar ratio. However, a wide range of molar ratios can be used to generate RNPs.

[0371] Recombinant adeno-associated virus (AAV) vectors can be used for delivery.In the art, the technique of producing rAAV particles is standard, which delivers the AAV genome packaged, including the polynucleotide to be delivered, rep and cap genes, and helper virus functions, to cells.The production of rAAV typically requires the following components to be present in a single cell (referred to herein as packaging cell): rAAV genome, AAV rep and cap genes that are separate from the rAAV genome (i.e., not present in the rAAV genome), and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype capable of deriving recombinant virus, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAV rh.74, which are different from the rAAV genome ITRs. The generation of pseudotyped rAAVs is disclosed, for example, in International Patent Application Publication No. WO 01 / 83692. See Table 3. [Table 3]

[0372] The method for producing packaging cells involves creating a cell line that stably expresses all of the components necessary to produce AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome can be integrated by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 20:2077-2081). 59:4661-4666) into bacterial plasmids. The packaging cell line can then be infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method utilizes adenovirus or baculovirus, rather than a plasmid, to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0373] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, pp. 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various techniques are described in Ratschin et al., Mol. Cell. Biol., 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol., 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988, Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828; U.S. Pat. No. 5,173,414; WO95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al. (1995), Vaccine, 13:1244-1250; Paul et al. (1993), Human Gene Therapy, 4:609-615; Clark et al. (1996), Gene Therapy, 3:1124-1132; U.S. Patent No. 5,786,211; U.S. Patent No. 5,8 No. 71,982; and U.S. Pat. No. 6,258,595.

[0374] The serotype of the AAV vector can be matched to the target cell type. For example, the following exemplary cell types can be transduced with the indicated AAV serotypes, among others. See Table 4. [Table 4]

[0375] Genetically modified cells

[0376] The term "genetically modified cell" refers to a cell containing at least one genetic modification introduced by genome editing (e.g., using a CRISPR / Cas system). In some ex vivo examples herein, the genetically modified cell may be a genetically modified progenitor cell. In some in vivo examples herein, the genetically modified cell may be a genetically modified muscle cell or a genetically modified muscle progenitor cell. Genetically modified cells containing an exogenous genome-targeting nucleic acid and / or an exogenous nucleic acid encoding a genome-targeting nucleic acid are contemplated herein.

[0377] The term "control-treated population" describes a population of cells treated with the same medium, viral induction, nucleic acid sequence, temperature, confluency, flask size, pH, etc., except for the addition of genome editing components. Any method known in the art, such as Western blot analysis for dystrophin protein or quantification of dystrophin mRNA, can be used to measure restoration of the dystrophin reading frame.

[0378] The term "isolated cell" refers to a cell that has been removed from an organism in which it is originally found, or the progeny of such a cell. Optionally, the cell can be cultured in vitro, for example, under defined conditions or in the presence of other cells. Optionally, the cell can later be introduced into a second organism, or reintroduced into the organism from which it was isolated (or into a cell that is its descendant).

[0379] The term "isolated population," with respect to an isolated population of cells, refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. Optionally, the isolated population can be a substantially pure population of cells compared to the heterogeneous population from which the cells are isolated or enriched. Optionally, the isolated population can be an isolated population of human progenitor cells, e.g., a substantially pure population of human progenitor cells compared to a heterogeneous population of cells that includes human progenitor cells and the cells from which the human progenitor cells are derived.

[0380] The term "substantially enhanced," with respect to a particular cell population, refers to a cell population in which the occurrence of a particular type of cell is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, at least 5000-fold, at least 20,000-fold, at least 100,000-fold, or more, relative to existing or baseline levels, depending on the desired level of such cells, e.g., to ameliorate DMD.

[0381] The term "substantially enriched" with respect to a particular cell population refers to a cell population that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more of the cells that make up the total cell population.

[0382] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, at least about 85%, at least about 90%, or at least about 95% pure with respect to the cells that make up the total cell population. That is, the term "substantially pure" or "essentially purified" with respect to a primary cell population refers to a cell population that contains less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1% of cells that are not primary cells as defined by the terms herein.

[0383] Differentiation of corrected iPSCs into Pax7+ muscle progenitor cells

[0384] Another step of the disclosed ex vivo method involves differentiating the corrected iPSCs into Pax7+ muscle progenitor cells. The differentiation step can be performed according to any method known in the art. For example, the differentiation step can be performed according to the method described in Chal, Oginuma et al., 201 This may involve contacting the genome-edited iPSCs with a specific media formulation containing a small molecule drug to differentiate them into Pax7+ muscle progenitor cells, as shown in Tapscott, Davis et al., 1988. Alternatively, iPSCs, myogenic progenitor cells, and cells of other lineages may be differentiated as described in Tapscott, Davis et al., 1988. ;Langen, Schols et al., 2003;Fujita, Endo et al., 2010;Xu, Tabebordbar et al., As shown in the methods of Shoji, Woltjen et al., 2013; Shoji, Woltjen et al., 2015, differentiation into muscle can be achieved using any one of several established methods involving transgene overexpression, serum deprivation, and / or small molecule drugs.

[0385] Implantation of Pax7+ myogenic progenitor cells into patients

[0386] Another step of the disclosed ex vivo method involves implanting the Pax7+ muscle progenitor cells into a patient. This implantation step can be accomplished using any implantation method known in the art. For example, the genetically modified cells can be injected directly into the patient's muscle.

[0387] Pharmaceutically acceptable carrier

[0388] The ex vivo methods of administering progenitor cells to a subject contemplated herein involve the use of therapeutic compositions comprising progenitor cells.

[0389] Therapeutic compositions may contain a physiologically tolerable carrier together with the cell composition, and optionally at least one additional bioactive agent, as described herein, dissolved or dispersed therein as an active ingredient. Optionally, the therapeutic composition, when administered to a mammal or human patient for therapeutic purposes, is substantially non-immunogenic, unless desired to be so.

[0390] Generally, the progenitor cells described herein can be administered as a suspension with a pharmaceutically acceptable carrier.Those skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition cannot contain buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the viability of the cells delivered to the subject.Cell-containing formulations can include, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and optionally, nutrients that maintain cell viability or enhance engraftment upon administration.Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the progenitor cells described herein using routine experimentation.

[0391] The cell compositions can also be emulsified and presented as liposomal compositions, provided that the emulsification procedure does not adversely affect the viability of the cells. The cells and any other active ingredients can be mixed with excipients that are pharmaceutically acceptable, compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0392] Additional agents contained in the cell composition may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0393] Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain no material other than the active ingredient and water, or buffers such as sodium phosphate, physiological saline, or phosphate-buffered saline at physiological pH values, or both.Furthermore, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.Liquid compositions can also contain liquid phases in addition to water and liquid phases other than water.Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.The amount of active compound used in the cell composition that is effective in treating a particular disorder or condition can depend on the nature of the disorder or condition and can be determined by standard clinical methods.

[0394] Dosage and Indications

[0395] In the context of transferring cells, e.g., progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, to produce a desired effect, the terms "administering," "introducing," and "implanting" are used interchangeably. Cells, e.g., progenitor cells, or their differentiated progeny, can be administered by any suitable route that results in delivery to a desired location in a subject, where at least some of the implanted cells or cellular components remain viable. Cell survival after administration to a subject can be as short as a few hours, e.g., 24 hours, to several days, to several years, or even as long as the patient's lifetime, i.e., long-term engraftment. For example, in some embodiments described herein, an effective amount of myogenic progenitor cells is administered via a systemic administration route, such as an intraperitoneal or intravenous route.

[0396] As used herein, the terms "individual," "subject," "host," and "patient" are used interchangeably and refer to any subject for whom diagnosis, treatment, or therapy is desired. In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0397] When administered prophylactically, the progenitor cells described herein can be administered to a subject prior to any symptoms of DMD, for example, prior to the onset of muscle mass loss. Thus, prophylactic administration of a population of muscle progenitor cells can be used to prevent DMD.

[0398] When administered therapeutically, the muscle progenitor cells can be administered at (or after) the onset of symptoms or signs of DMD, for example, at the onset of muscle mass loss.

[0399] The muscle progenitor cell population administered according to the methods described herein can include allogeneic muscle progenitor cells obtained from one or more donors. "Allogeneic" refers to muscle progenitor cells or biological samples containing muscle progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical. For example, the muscle progenitor cell population administered to a subject can be derived from one or more unrelated donor subjects or from one or more non-identical sibling species. In some cases, syngeneic muscle progenitor cell populations, such as populations obtained from genetically identical animals or identical twins, can also be used. The muscle progenitor cells can also be autologous, i.e., the muscle progenitor cells are obtained or isolated from a subject and administered to the same subject, i.e., the donor and recipient are the same.

[0400] The term "effective amount" refers to the amount of a population of progenitor cells or their progeny required to prevent or alleviate at least one or more signs or symptoms of DMD, and relates to the amount of a composition sufficient to produce a desired effect, e.g., to treat a subject with DMD. Thus, the term "therapeutically effective amount" refers to the amount of progenitor cells or a composition comprising progenitor cells that, when administered to a typical subject, such as a subject with or at risk of DMD, is sufficient to promote a particular effect. An effective amount would also include an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It is understood that an appropriate "effective amount" for any given case can be determined by one of ordinary skill in the art using routine experimentation.

[0401] For use in the various embodiments described herein, an effective amount of progenitor cells is at least 10 progenitor cells. 2 At least 5 × 10 progenitor cells 2 At least 10 progenitor cells 3 At least 5 × 10 progenitor cells 3At least 10 progenitor cells 4 At least 5 × 10 progenitor cells 4 At least 10 progenitor cells 5 At least 2 x 10 progenitor cells 5 At least 3 x 10 progenitor cells 5 At least 4 x 10 progenitor cells 5 At least 5 × 10 progenitor cells 5 At least 6 x 10 progenitor cells 5 At least 7 x 10 progenitor cells 5 At least 8 x 10 progenitor cells 5 , at least 9 × 10 progenitor cells 5 At least 1 x 10 progenitor cells 6 At least 2 x 10 progenitor cells 6 At least 3 x 10 progenitor cells 6 At least 4 x 10 progenitor cells 6 At least 5 × 10 progenitor cells 6 At least 6 x 10 progenitor cells 6 At least 7 x 10 progenitor cells 6 At least 8 x 10 progenitor cells 6 , at least 9 × 10 progenitor cells 6 The progenitor cells may be derived from one or more donors or from an autologous source. In some examples described herein, the progenitor cells may be expanded in culture before being administered to a subject in need thereof.

[0402] Gradually increasing the level of functional dystrophin expressed in cells of patients with DMD can be beneficial for ameliorating one or more symptoms of the disease, increasing long-term survival, and / or reducing side effects associated with other treatments. When such cells are administered to a human patient, the presence of muscle progenitor cells that result in elevated levels of functional dystrophin is beneficial. In some cases, effective treatment of a subject results in at least about 3%, 5%, or 7% functional dystrophin relative to the total dystrophin in the treated subject. In some cases, functional dystrophin will be at least about 10% of the total dystrophin. In some cases, functional dystrophin will be at least about 20%-30% of the total dystrophin. Similarly, because normalized cells have a selective advantage over diseased cells in some situations, the introduction of even a relatively limited subpopulation of cells with significantly elevated levels of functional dystrophin can still be beneficial in a variety of patients. However, even a small level of muscle progenitor cells can be beneficial for improving one or more aspects of DMD in patients with increased functional dystrophin levels.In some cases, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the muscle progenitor cells in patients receiving such cells produce high levels of functional dystrophin.

[0403] "Administered" refers to delivery of the progenitor cell composition to a subject by a method or route that results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in the subject, i.e., administration that results in delivery to a desired location in the subject, with at least a portion of the delivered composition, i.e., at least 1 x 10 cells. 4The cells can be administered by administration to deliver them to the desired site over a period of time. Administration methods include injection, infusion, drip infusion, or oral administration. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some cases, the route is intravenous. To deliver cells, administration can be performed by injection or infusion.

[0404] The cells are administered systemically. The phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to administration of a population of progenitor cells other than directly to a target site, tissue, or organ, but instead such that it enters the subject's circulatory system and is therefore subject to metabolic and other similar processes.

[0405] Those skilled in the art can determine the efficacy of a treatment, including a composition for treating DMD. However, by way of example only, a treatment is considered "effective" if any one or all of the signs or symptoms of functional dystrophin levels are beneficially altered (e.g., increased by at least 10%), or other clinically acceptable symptoms or markers of the disease are improved or alleviated. Efficacy can also be measured by the non-deterioration of an individual (e.g., reduced muscle mass loss, or the progression of the disease is stopped or at least slowed), as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing a decline in symptoms; and (3) preventing or reducing the likelihood of the onset of symptoms.

[0406] Treatment according to the present disclosure can alleviate one or more symptoms associated with DMD by increasing the amount of functional dystrophin in individuals.Early symptoms typically associated with DMD include, for example, delayed walking, calf muscle hypertrophy (due to scar tissue), and frequent falls.As the disease progresses, children become wheelchair-bound due to muscle loss and pain.Disease can be fatal due to cardiac and / or respiratory complications.

[0407] kit

[0408] The present disclosure provides kits for carrying out the methods described herein. The kits may include one or more of the following: a genome-targeting nucleic acid, a polynucleotide encoding the genome-targeting nucleic acid, a site-directed polypeptide, a polynucleotide encoding the site-directed polypeptide, and / or any nucleic acid or proteinaceous molecule required to carry out an embodiment of the method described herein, or any combination thereof.

[0409] The kit may include (1) a vector containing a nucleotide sequence encoding a genome-targeting nucleic acid, (2) a site-directed polypeptide or a vector containing a nucleotide sequence encoding a site-directed polypeptide, and (3) reagents for repairing and / or diluting the vector and / or polypeptide.

[0410] The kit may include (1) a vector containing (i) a nucleotide sequence encoding a genome-targeting nucleic acid and (ii) a nucleotide sequence encoding a site-specific polypeptide, and (2) reagents for repairing and / or diluting the vector.

[0411] In some of the kits, the kit may include a single-molecule genome-targeting guide nucleic acid. In any of the above kits, the kit may include a double-molecule genome-targeting nucleic acid. In any of the kits, the kit may include two or more double-molecule guides or single-molecule guides. The kit may include a vector encoding the nucleic acid-targeting nucleic acid.

[0412] In any of the kits, the kit may further include a polynucleotide to be inserted to effect the desired genetic modification.

[0413] The components of the kit may be in separate containers or may be combined in a single container.

[0414] Any kit may further comprise one or more additional reagents, where such additional reagents are selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, washing buffers, control reagents, control vectors, control RNA polynucleotides, reagents for in vitro production of polypeptides from DNA, adapters for sequencing, etc. The buffer may be a stabilization buffer, repair buffer, dilution buffer, etc. The kit may also comprise one or more components that can be used to facilitate or enhance on-target binding or endonuclease cleavage of DNA, or to improve targeting specificity.

[0415] In addition to the components mentioned above, the kit may further include instructions for practicing a method using the components of the kit. The instructions for practicing the method may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert, in the labeling of the container (i.e., associated with the packaging or sub-packaging) of the kit or its components, etc. The instructions may be present as an electronically stored data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this case is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0416] Guide RNA formulations

[0417] Guide RNAs of the present disclosure can be formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the particular mode of administration and dosage form. Guide RNA compositions can be formulated to achieve a physiologically compatible pH, ranging from about pH 3 to about pH 11, or from about pH 3 to about pH 7, depending on the formulation and route of administration. Optionally, the pH can be adjusted to a range from about pH 5.0 to about pH 8. Optionally, the composition can comprise a therapeutically effective amount of at least one compound described herein in combination with one or more pharmaceutically acceptable excipients. Optionally, the composition can include a combination of compounds described herein, a second active ingredient (e.g., and without limitation, an antibacterial or antimicrobial agent) useful in treating or preventing bacterial growth, or a combination of reagents of the present disclosure.

[0418] Suitable excipients include carrier molecules comprising large, slowly metabolized macromolecules, such as, for example, proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients can include antioxidants (for example, and without limitation, ascorbic acid), chelating agents (for example, and without limitation, EDTA), carbohydrates (for example, and without limitation, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example, and without limitation, oil, water, saline, glycerol, and ethanol), humectants or emulsifiers, pH buffering substances, and the like.

[0419] Other possible treatments

[0420] Gene editing can be performed using nucleases engineered to target specific sequences. Currently, there are four major types of nucleases: meganucleases and their derivatives, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 nuclease system. Notably, the specificity of ZFNs and TALENs is mediated by protein-DNA interactions, whereas RNA-DNA interactions primarily guide Cas9. Nuclease platforms vary in design difficulty, targeting density, and mode of action. Cas9 cleavage also requires a neighboring motif, the PAM, which differs among different CRISPR systems. Cas9 from Streptococcus pyogenes cleaves using the PAM NGG, while CRISPR from Neisseria meningitidis can cleave at sites with PAMs including NNNNGATT, NNNNGTTTT, and NNNNGCTT. Several other Cas9 orthologs target alternative PAMs and adjacent protospacers.

[0421] In the method of the present disclosure, CRISPR endonuclease such as Cas9 can be used.But the teachings described herein, such as therapeutic target site, can also be applied to other forms of endonuclease, such as ZFN, TALEN, HE or MegaTAL, and can also be applied to the use of combinations of nucleases.But, in order to apply the teachings of the present disclosure to such endonuclease, it will be necessary to specifically engineer the protein that is directed to specific target site.

[0422] Additional binding domains can be fused to the Cas9 protein to increase specificity. The target sites of these constructs map to the identified gRNA-directed site but will require additional binding motifs, such as for zinc finger domains. In the case of Mega-TALs, meganucleases can be fused to TALE DNA-binding domains. Meganuclease domains can increase specificity and effect cleavage. Similarly, inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain, which requires an sgRNA / Cas9 target site and adjacent binding sites for the fused DNA-binding domain. This will likely require some proteolytic engineering of dCas9 in addition to catalytic inactivation to reduce binding without additional binding sites.

[0423] Zinc finger nuclease

[0424] Zinc finger nucleases (ZFNs) are modular proteins composed of an engineered zinc finger DNA-binding domain linked to the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to cognate target "half-site" sequences on opposite DNA strands with precise spacing between them to allow the formation of catalytically active FokI dimers. Dimerization of the FokI domain, which has no sequence specificity by itself, creates a DNA double-strand break between the ZFN half-sites as the initiating step in genome editing.

[0425] The DNA-binding domain of each ZFN typically consists of three to six zinc fingers with a Cys2-His2 architecture, each of which primarily recognizes a triplet of nucleotides on one strand of the target DNA sequence, although cross-interactions with the fourth nucleotide can also be important. Altering the amino acids of a finger at positions that make key contact points with DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein selectively recognizes a 12-bp target sequence; however, triplet preference can be influenced to varying degrees by neighboring fingers, and the target sequence is a composite of the triplet preferences contributed by each finger. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address simply by modifying individual fingers, although this requires considerable skill to achieve. Most applications of ZFNs use proteins with four to six fingers, each recognizing 12 to 18 bp. Thus, a pair of ZFNs will typically recognize a combination of 24-36 bp target sequences, without the typically 5-7 bp spacer between half-sites. Binding sites can be further separated by a large spacer, including 15-17 bp. Target sequences of this length are likely to be unique within the human genome, assuming repeat sequences or gene homologs are excluded during the design process. However, ZFN protein-DNA interactions are not absolute in their specificity, and off-target binding and cleavage events can occur as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" mutants, also known as obligate heterodimer mutants, that can dimerize only with each other and not with themselves. Forcing obligate heterodimers prevents the formation of homodimers. This greatly enhances the specificity of ZFNs, as well as any other nucleases that employ these FokI mutants.

[0426] A variety of ZFN-based systems have been described in the art, modifications of which are regularly reported, and numerous references describe the rules and parameters used to guide the design of ZFNs (e.g., Segal et al., Proc Natl Acad Sci USA, Vol. 96). (No. 6): pp. 2758-63 (1999); Dreier B et al., J Mol Biol., vol. 303 (No. 4): pp. 489-502 (2000); Liu Q et al., J Biol Chem., vol. 277 (No. 6): pp. 3850-6 (2002); Dreier et al., J Biol Chem., vol. 280 (No. 42): 35588-97 (2005); and Dreier et al., J Biol Chem., 276(31):29466-78 (2001).

[0427] Transcription activator-like effector nucleases (TALENs)

[0428] TALENs are another modular nuclease format. Similar to ZFNs, they incorporate an engineered DNA-binding domain linked to a FokI nuclease domain, allowing a pair of TALENs to operate in tandem to achieve targeted DNA cleavage. The primary difference between TALENs and ZFNs is the nature of the DNA-binding domain and the associated target DNA sequence recognition properties. The DNA-binding domain of TALENs is derived from TALE proteins, originally described in Xanthomonas sp., a bacterial plant pathogen. TALEs are tandem arrays of 33-35 amino acid repeats, each recognizing a single base pair within a target DNA sequence, typically up to 20 bp in length, with the total target sequence length being up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable residue (RVD), which contains only two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This constitutes a much simpler recognition code than that of zinc fingers and therefore represents an advantage over the latter for nuclease design. However, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs also benefit from the use of obligate heterodimer mutants of the FokI domain to reduce off-target activity.

[0429] Additional mutants of the FokI domain that inactivate their catalytic function have also been created. If one member of a TALEN or ZFN pair contains an inactive FokI domain, only single-strand DNA breaks (nicking) will occur at the target site, rather than DSBs. The result is equivalent to using a "nickase" mutant of CRISPR / Cas9 / Cpf1 in which one of the Cas9 cleavage domains is inactivated. DNA nicks can be used to drive genome editing via HDR, but with lower efficiency than DSBs. A major advantage is that off-target nicks are repaired quickly and accurately, unlike DSBs, which are prone to misrepair mediated by NHEJ.

[0430] Various TALEN-based systems have been described in the art, and modifications thereof are regularly reported (see, e.g., Boch, Science, 326(5959):1509-1510). 12 (2009); Mak et al., Science, 335 (Issue 6069): 716-9 (2012); and Moscou et al., Science, 326 (Issue 5959): 1501 (2009 The use of TALENs based on the "GoldenGate" platform or cloning scheme has been described by several groups (e.g., Cermak et al., Nucleic Acids Res., 39(12):e82 (2011); Li et al., Nucleic Acids Res., 39(14):6315-25 (2011); Weber et al., PLoS One., 6(2):e16765 (2011); Wang et al., J Genet Genomics, 41(6):339-47, Epub, May 17, 2014 (2011)). 4 years); and Cermak T et al., Methods Mol Biol., 1239:133-59 (2015).

[0431] Homing endonucleases Homing endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity, often at unique sites within the genome. There are at least six known families of HEs, classified by their structure, including LAGLIDADG (SEQ ID NO: 1,410,474), GIY-YIG, His-Cis box, HNH, PD-(D / E)xK, and Vsr-like, which are derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages. Similar to ZFNs and TALENs, HEs can be used to create DSBs at target loci as the first step in genome editing. In addition, some natural and engineered HEs cleave only a single strand of DNA, thereby functioning as site-specific nickases. The large target sequences of HEs and the specificity they confer make them attractive candidates for creating site-specific DSBs.

[0432] A variety of HE-based systems have been described in the art, and modifications thereof are regularly reported (e.g., Steentoft et al., Glycobiology, 24(8):663-80). (2014); Belfort and Bonocora, Methods Mol Biol., 1123:1-26 (2014); Hafez and Hausner, Genome, 55(8):553-69 (2012); and the reviews by Hafez and Hausner, Genome, 55(8):553-69 (2012); and the references cited therein).

[0433] MegaTAL / Tev-mTALEN / MegaTev

[0434] As further examples of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms use fusions of a TALE DNA-binding domain with a catalytically active HE, exploiting both the fine-tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of the HE (e.g., Boissel et al., NAR 42:2591-2601 (2014); Kleinstiver et al., G3 4:1155-65). (2014); and Boissel and Scharenberg, Methods Mol. Biol., 1239:171-96 (2015).

[0435] In a further variation, the MegaTev architecture is a fusion of Meganuclease (Mega) with I-TevI ​​(Tev), a nuclease domain derived from the GIY-YIG homing endonuclease. The two active sites are located approximately 30 bp apart in the DNA substrate, generating two DSBs with incompatible sticky ends (see, e.g., Wolfs et al., NAR, 42, 8816-29 (2014)). It is expected that other combinations of existing nuclease-based approaches will evolve and be useful in achieving the targeted genome modifications described herein.

[0436] dCas9-FokI or dCpf1-Fok1 and other nucleases

[0437] The structural and functional characteristics of the nuclease platform described above are combined to provide a new approach to genome editing that can potentially overcome some of its inherent drawbacks. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to create DSBs. Targeting specificity is driven by a 20- or 24-nucleotide sequence in the guide RNA that undergoes Watson-Crick base pairing with the target DNA (in the case of Cas9 derived from S. pyogenes, two additional bases in the adjacent PAM sequence, NAG or NGG, are added). Although such sequences are long enough to be unique within the human genome, the specificity of RNA / DNA interactions is not absolute, and in some cases, significant promiscuity is tolerated, especially at the 5' end of the target sequence, effectively reducing the number of bases that drive specificity. One solution to this problem has been to fuse the FokI domain to the inactivated Cas9 instead of completely inactivating the catalytic function of Cas9 or Cpf1 (while retaining only the RNA-guided DNA-binding function) (see, e.g., Tsai et al., Nature Biotech. 32:569-76 (2014); and Guilinger et al., Nature Biotech. 32:577-82 (2014)). Because FokI must dimerize to become catalytically active, two guide RNAs are required to tether the two FokI fusions in close proximity to form dimers and cleave DNA. This essentially doubles the number of bases in the target site combination, thereby increasing the targeting stringency of CRISPR-based systems.

[0438] As a further example, fusion of a TALE DNA-binding domain to a catalytically active HE, such as I-TevI, takes advantage of both the fine-tunable DNA binding and specificity of the TALE, as well as the cleavage sequence specificity of I-TevI, in the hopes that off-target cleavage may be further reduced.

[0439] Methods and Compositions of the Invention

[0440] Accordingly, the present disclosure particularly relates to the following non-limiting inventions: In a first method, Method 1, the present disclosure provides a method for editing a dystrophin gene in a human cell by genome editing, comprising the step of introducing into the human cell one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, which SSBs or DSBs result in the permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0441] In another method, Method 2, the present disclosure provides a method for editing a dystrophin gene in a human cell by genome editing as provided in Method 1, wherein the human cell is a muscle cell or a muscle progenitor cell.

[0442] In another method, Method 3, the present disclosure provides an ex vivo method for treating a patient with Duchenne muscular dystrophy (DMD), comprising: i) creating DMD patient-specific induced pluripotent stem cells (iPSCs); ii) editing the iPSCs within or near the dystrophin gene; iii) differentiating the genome-edited iPSCs into Pax7+ muscle progenitor cells; and iv) implanting the Pax7+ muscle progenitor cells into the patient.

[0443] In another method, Method 4, the present disclosure provides an ex vivo method for treating a patient with DMD as provided in Method 3, wherein the creating step includes: a) isolating somatic cells from the patient; and b) introducing a set of pluripotency-associated genes into the somatic cells to induce the somatic cells to become pluripotent stem cells.

[0444] In another method, Method 5, the present disclosure provides an ex vivo method for treating a patient with DMD as provided in Method 4, wherein the somatic cells are fibroblasts.

[0445] In another method, Method 6, the present disclosure provides an ex vivo method for treating a patient with DMD as provided in Methods 4 and 5, wherein the set of pluripotency-associated genes is one or more of genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

[0446] In another method, Method 7, the present disclosure provides an ex vivo method for treating a patient with DMD, as provided in any one of Methods 3-6, wherein the editing step includes introducing one or more deoxyribonucleic acid (DNA) endonucleases into the iPSCs to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, the SSBs or DSBs resulting in a permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0447] In another method, Method 8, the present disclosure provides an ex vivo method for treating a patient with DMD as provided in any one of Methods 3-7, wherein the differentiating step includes one or more of the following: contacting the genome-edited iPSCs with a specific media formulation comprising a small molecule drug; overexpression of a transgene; or serum deprivation, so as to differentiate the genome-edited iPSCs into Pax7+ muscle progenitor cells.

[0448] In another method, Method 9, the present disclosure provides an ex vivo method for treating a patient with DMD, as provided in any one of Methods 3 to 8, wherein the implanting step includes implanting Pax7+ muscle progenitor cells into the patient by local injection into the desired muscle.

[0449] In another method, Method 10, the present disclosure provides an in vivo method for treating a patient with DMD, the method comprising editing a dystrophin gene in the patient's cells.

[0450] In another method, Method 11, the present disclosure presents an in vivo method for treating a patient with DMD as presented in Method 10, wherein the editing step includes introducing into the patient's cells one or more deoxyribonucleic acid (DNA) endonucleases to create one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the dystrophin gene, the SSBs or DSBs resulting in a permanent deletion, insertion, or replacement of one or more exons, or splice acceptor or donor sites of an aberrant intron, within or near the dystrophin gene, thereby restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0451] In another method, Method 12, the present disclosure provides an in vivo method for treating a patient with DMD as provided in Method 11, wherein the cells are muscle cells or muscle progenitor cells.

[0452] In another method, Method 13, the disclosure provides an in vivo method for treating a patient with DMD as provided in any one of Methods 1, 7, and 11, wherein the one or more DNA endonucleases are selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6 , Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonucleases; homologs thereof, recombinant forms of these naturally occurring molecules, codon-optimized forms thereof, modified forms thereof, and combinations thereof are presented.

[0453] In another method, Method 14, the present disclosure provides the method provided in Method 13, including introducing into the cell one or more polynucleotides encoding one or more DNA endonucleases.

[0454] In another method, Method 15, the present disclosure provides the method provided in Method 13, including introducing into the cell one or more ribonucleic acids (RNAs) encoding one or more DNA endonucleases.

[0455] In another method, Method 16, the present disclosure provides the methods provided in Methods 14 and 15, wherein the one or more polynucleotides or one or more RNAs are one or more modified polynucleotides or one or more modified RNAs.

[0456] In another method, method 17, the present disclosure provides the method as provided in method 13, wherein the one or more DNA endonucleases are one or more proteins or polypeptides.

[0457] In another method, Method 18, the present disclosure provides the method provided in any one of Methods 1-17, further comprising introducing one or more guide ribonucleic acids (gRNAs) into the cell.

[0458] In another method, Method 19, the present disclosure provides the method as provided in Method 18, wherein one or more gRNAs are single-molecule guide RNAs (sgRNAs).

[0459] In another method, Method 20, the present disclosure provides the methods provided in Methods 18 and 19, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0460] In another method, Method 21, the present disclosure provides a method as provided in any one of Methods 18-20, wherein one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs.

[0461] In another method, Method 22, the present disclosure provides the method of any one of Methods 1 to 21, further comprising introducing into the cell a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene or cDNA.

[0462] In another method, Method 23, the disclosure provides a method for determining whether at least a portion of a wild-type dystrophin gene or cDNA is selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, exon 8 exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, an intron region, a synthetic intron region, a fragment, a combination thereof, or at least a portion of the entire dystrophin gene or cDNA.

[0463] In another method, Method 24, the disclosure provides a method comprising: providing a dystrophin gene or cDNA comprising at least a portion of the dystrophin gene or cDNA comprising exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42 , exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

[0464] In another method, Method 25, the present disclosure provides the method of any one of Methods 22-24, wherein the donor template is a single-stranded or double-stranded polynucleotide.

[0465] In another method, Method 26, the present disclosure provides the method of any one of Methods 1, 7, and 11, further comprising introducing into the cell one or more guide ribonucleic acids (gRNAs), wherein the one or more DNA endonucleases are one or more Cas9 or Cpfl endonucleases that generate a pair of single-strand breaks (SSBs) or double-strand breaks (DSBs), where a first SSB or DSB cleaves at the 5' locus and a second SSB or DSB cleaves at the 3' locus, that result in the permanent deletion or replacement of one or more exons, or splice acceptor or donor sites of the aberrant intron, between the 5' and 3' loci within or near the dystrophin gene, thereby restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0466] In another method, Method 27, the present disclosure provides the method as provided in Method 26, wherein one gRNA creates a pair of SSBs or DSBs.

[0467] In another method, Method 28, the present disclosure provides the method as provided in Method 26, wherein one gRNA includes a spacer sequence that is complementary to the 5' locus, the 3' locus, or a segment between the 5' locus and the 3' locus.

[0468] In another method, Method 29, the present disclosure provides the method as provided in Method 26, comprising a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence that is complementary to a segment of the 5' locus, and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3' locus.

[0469] In another method, Method 30, the present disclosure provides any of the methods provided in Methods 26-29, wherein the one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).

[0470] In another method, Method 31, the present disclosure provides a method as provided in Methods 26-30, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0471] In another method, Method 32, the present disclosure provides a method as provided in any one of Methods 26-31, wherein one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs.

[0472] In another method, Method 33, the present disclosure provides any one of Methods 26-32, wherein there is a deletion of chromosomal DNA between the 5' and 3' loci.

[0473] In another method, Method 34, the present disclosure provides the method of any one of Methods 26-33, wherein the deletion is a single exon deletion.

[0474] In another method, Method 35, the present disclosure provides the method as provided in Method 34, wherein the single exon deletion is a deletion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, or exon 53.

[0475] In another method, Method 36, the present disclosure provides the method as provided in Methods 34 or 35, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53.

[0476] In another method, Method 37, the present disclosure provides any one of Methods 34-36, wherein the 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53.

[0477] In another method, Method 38, the present disclosure provides any one of Methods 34-37, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, and exon 53, and the 3' locus is proximal to the 3' boundary of such a single exon.

[0478] In another method, Method 39, the present disclosure provides any one of Methods 36-38, wherein the proximity to the exon boundary includes a splice donor and acceptor around the adjacent intron.

[0479] In another method, Method 40, the present disclosure provides the method of any one of Methods 26-33, wherein the deletion is a multi-exon deletion.

[0480] In another method, method 41, the present disclosure provides the method as provided in method 40, wherein the multi-exon deletion is a deletion of exons 45-53 or exons 45-55.

[0481] In another method, Method 42, the present disclosure provides any one of Methods 40-41, wherein the 5' locus is proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

[0482] In another method, Method 43, the present disclosure provides any one of Methods 40-42, wherein the 3' locus is proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

[0483] In another method, Method 44, the present disclosure provides a method as provided in any one of Methods 40-43, wherein the 5' locus is proximal to the 5' boundary of a plurality of exons selected from the group consisting of exons 45-53 and exons 45-55, and the 3' locus is proximal to the 3' boundary of these exons.

[0484] In another method, method 45, the present disclosure provides any one of methods 42-44, wherein the proximity to the exon boundary includes a splice donor and acceptor around the adjacent intron.

[0485] In another method, Method 46, the present disclosure provides a method as provided in any one of Methods 26-32, wherein there is a chromosomal DNA replacement between the 5' locus and the 3' locus.

[0486] In another method, Method 47, the present disclosure provides the method provided in any one of Methods 26-32 and 46, wherein the replacement is a single exon replacement.

[0487] In another method, Method 48, the present disclosure provides any one of Methods 26-32 and 46-47, wherein the single exon replacement is a replacement of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70.

[0488] In another method, Method 49, the disclosure provides any one of Methods 47-48, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70.

[0489] In another method, method 50, the disclosure provides any one of methods 47-49, wherein the 3' locus is proximal to the 3' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70.

[0490] In another method, Method 51, the present disclosure provides a method as provided in any one of Methods 47-50, wherein the 5' locus is proximal to the 5' boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70, and the 3' locus is proximal to the 3' boundary of such a single exon.

[0491] In another method, Method 52, the present disclosure provides any one of Methods 49-51, wherein the proximity to the exon boundary includes a splice donor and acceptor around an adjacent intron or adjacent exon.

[0492] In another method, method 53, the present disclosure provides the method of any one of methods 26-32 or 46, wherein the replacement is a multi-exon replacement.

[0493] In another method, method 54, the present disclosure provides the method as provided in method 53, wherein the multi-exon replacement is a replacement of exons 45-53 or exons 45-55.

[0494] In another method, Method 55, the present disclosure provides any one of Methods 53-54, wherein the 5' locus is proximal to the 5' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

[0495] In another method, Method 56, the present disclosure provides any one of Methods 53-55, wherein the 3' locus is proximal to the 3' boundary of multiple exons selected from the group consisting of exons 45-53 and exons 45-55.

[0496] In another method, Method 57, the present disclosure provides a method as provided in any one of Methods 53-56, wherein the 5' locus is proximal to the 5' boundary of a plurality of exons selected from the group consisting of exons 45-53 and exons 45-55, and the 3' locus is proximal to the 3' boundary of these exons.

[0497] In another method, Method 58, the present disclosure provides any one of Methods 55-57, wherein the proximity to the exon boundary includes a splice donor and acceptor around the adjacent intron.

[0498] In another method, Method 59, the present disclosure provides any one of Methods 46-58, further comprising introducing into the cell a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene or cDNA, wherein the replacement is by homology-directed repair (HDR).

[0499] In another method, method 60, the disclosure provides a method for determining whether at least a portion of a wild-type dystrophin gene or cDNA is selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, exon 8 exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, an intron region, a synthetic intron region, a fragment, a combination thereof, or at least a portion of the entire dystrophin gene or cDNA.

[0500] In another method, Method 61, the disclosure provides a method for determining whether at least a portion of a wild-type dystrophin gene or cDNA is selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42 , exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, intron regions, synthetic intron regions, fragments, combinations thereof, or the entire dystrophin gene or cDNA.

[0501] In another method, Method 62, the present disclosure provides the method of any one of Methods 1, 7, or 11, further comprising introducing into the cell a guide ribonucleic acid (gRNA) and a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene, wherein the one or more DNA endonucleases are one or more Cas9 or Cpfl endonucleases that create a single-strand break (SSB) or double-strand break (DSB) at a locus within or near the dystrophin gene that facilitates insertion of new sequences derived from the polynucleotide donor template into chromosomal DNA at the locus, which SSB or DSB results in permanent insertion or correction of one or more exons, or aberrant intronic splice acceptor or donor sites within or near the dystrophin gene, restoring the dystrophin reading frame and restoring activity of the dystrophin protein, and wherein the gRNA comprises a spacer sequence that is complementary to a segment of the locus.

[0502] In another method, method 63, the disclosure further comprises introducing into a cell one or more guide ribonucleic acids (gRNAs) and a polynucleotide donor template comprising at least a portion of a wild-type dystrophin gene, wherein one or more DNA endonucleases create a pair of single-strand breaks (S1, S2) within or near the dystrophin gene, the first at the 5' locus and the second at the 3' locus, that facilitate insertion of new sequences derived from the polynucleotide donor template into chromosomal DNA between the 5' and 3' loci. 12. The method of any one of methods 1, 7, or 11, wherein the one or more Cas9 or Cpfl endonucleases create an SSB or double-strand break (DSB) that results in the permanent insertion or correction of one or more exons, or aberrant intron splice acceptor or donor sites, between the 5' and 3' loci within or near the dystrophin gene, restoring the dystrophin reading frame and restoring the activity of the dystrophin protein.

[0503] In another method, method 64, the present disclosure provides the method provided in method 63, wherein one gRNA creates a pair of SSBs or DSBs.

[0504] In another method, Method 65, the present disclosure provides the method as provided in Method 63, wherein one gRNA includes a spacer sequence that is complementary to the 5' locus, the 3' locus, or a segment between the 5' locus and the 3' locus.

[0505] In another method, Method 66, the present disclosure provides the method provided in Method 63, comprising a first gRNA and a second gRNA, wherein the first gRNA comprises a spacer sequence that is complementary to a segment of the 5' locus, and the second gRNA comprises a spacer sequence that is complementary to a segment of the 3' locus.

[0506] In another method, method 67, the present disclosure provides the method as provided in method 62 or 63, wherein the one or more gRNAs are one or more single molecule guide RNAs (sgRNAs).

[0507] In another method, Method 68, the present disclosure provides the method provided in Methods 62-63 or 67, wherein the one or more gRNAs or one or more sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0508] In another method, Method 69, the present disclosure provides a method as provided in any one of Methods 62-63 or 67-68, wherein one or more DNA endonucleases are pre-complexed with one or more gRNAs or one or more sgRNAs.

[0509] In another method, method 70, the present disclosure provides the method of any one of methods 62-69, wherein the insertion is a single exon insertion.

[0510] In another method, method 71, the present disclosure provides the method as provided in method 70, wherein the single exon insertion is an insertion of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 70.

[0511] In another method, Method 72, the present disclosure provides any one of Methods 70-71, wherein the locus, 5' locus or 3' locus is proximal to the boundary of a single exon selected from the group consisting of exon 2, exon 8, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53 and exon 70.

[0512] In another method, method 73, the present disclosure provides the method as provided in method 72, wherein the proximity to the exon boundary includes a splice donor and acceptor around an adjacent intron or adjacent exon.

[0513] In another method, method 74, the present disclosure provides the method of any one of methods 62-69, wherein the insertion is a multi-exon insertion.

[0514] In another method, method 75, the present disclosure provides the method as provided in method 74, wherein the multi-exon insertion is an insertion of exons 45-53 or exons 45-55.

[0515] In another method, Method 76, the present disclosure provides a method as provided in any one of Methods 74-75, wherein the locus, the 5' locus, or the 3' locus is proximal to a boundary of a multi-exon selected from the group consisting of exons 45-53 or exons 45-55.

[0516] In another method, method 77, the present disclosure provides the method as provided in method 76, wherein the proximity to the exon boundary includes a splice donor and acceptor around the adjacent intron.

[0517] In another method, Method 78, the disclosure provides a method for determining whether at least a portion of a wild-type dystrophin gene or cDNA is selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43 , exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, an intron region, a synthetic intron region, a fragment, a combination thereof, or at least a portion of the entire dystrophin gene or cDNA.

[0518] In another method, method 79, the disclosure provides a method for determining whether at least a portion of a wild-type dystrophin gene or cDNA is selected from exon 1, exon 2, exon 3, exon 4, exon 5, exon 6,

[0519] Exon 7, Exon 8, Exon 9, Exon 10, Exon 11, Exon 12, Exon 13, Exon 14, Exon 15, Exon 16, Exon 17, Exon 18, Exon 19, Exon 20, Exon 21, Exon 22, Exon 23, Exon 24, Exon 25, Exon 26, Exon 27, Exon 28, Exon 29, Exon 30, Exon 31, Exon 32, Exon 33, Exon 34, Exon 35, Exon 36, Exon 37, Exon 38, Exon 39, Exon 40, Exon 41, Exon 42, Exon 43, Exon 44, Exon 45, Exon 46, Exon 47, Exon 48, Exon exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, an intron region, a synthetic intron region, a fragment, a combination thereof, or the entire dystrophin gene or cDNA.

[0520] In another method, method 80, the present disclosure provides the method provided in any one of methods 62-79, wherein the insertion is by homology-guided repair (HDR).

[0521] In another method, method 81, the present disclosure provides any one of methods 62-80, wherein the donor template is a single-stranded or double-stranded polynucleotide.

[0522] In another method, Method 82, the present disclosure provides a method as provided in any one of Methods 26-81, wherein the mRNA, gRNA, and donor template for Cas9 or Cpf1 are each formulated into separate lipid nanoparticles, or all of these are co-formulated into lipid nanoparticles.

[0523] In another method, Method 83, the present disclosure provides a method as provided in any one of Methods 26-81, wherein the Cas9 or Cpf1 mRNA is formulated into a lipid nanoparticle, and both the gRNA and the donor template are delivered to the cell by an adeno-associated virus (AAV) vector.

[0524] In another method, Method 84, the present disclosure provides a method as provided in any one of Methods 26-81, wherein the Cas9 or Cpf1 mRNA is formulated into lipid nanoparticles, the gRNA is delivered to the cell by electroporation, and the donor template is delivered to the cell by an adeno-associated virus (AAV) vector.

[0525] In another method, Method 85, the present disclosure provides the method of any one of Methods 1 to 84, wherein the dystrophin gene is located on chromosome X: 31,117,228-33,344,609 (Genome Reference Consortium: GRCh38 / hg38).

[0526] In a first composition, Composition 1, the present disclosure provides one or more guide ribonucleic acids (gRNAs) for editing a dystrophin gene in cells derived from a patient with Duchenne muscular dystrophy (DMD), the one or more gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 1 to 1,410,472 of the Sequence Listing.

[0527] In another composition, Composition 2, the present disclosure provides one or more gRNAs of Composition 1, wherein the one or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).

[0528] In another composition, Composition 3, the present disclosure provides one or more gRNAs or sgRNAs of Composition 1 or 2, wherein the one or more gRNAs or sgRNAs are one or more modified gRNAs or one or more modified sgRNAs.

[0529] definition

[0530] The terms "comprising" or "comprising" are used in reference to compositions, methods, and their respective components that are essential to the invention, but are open to the inclusion of unspecified elements, whether essential or dispensable.

[0531] The term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic, novel, or functional characteristics of that embodiment of the invention.

[0532] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in that description of the embodiment.

[0533] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0534] Any numerical range recited herein describes all subranges of the same numerical precision (i.e., having the same number of digits as the specified number) that are encompassed within the recited range. For example, a recited range of "1.0 to 10.0" describes all subranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 10.0, such as "2.4 to 7.6," even if a range such as "2.4 to 7.6" is not explicitly recited in the body of the specification. Accordingly, applicant reserves the right to amend this specification, including the claims, to explicitly recite any subrange of the same numerical precision that is encompassed within a range explicitly recited herein. All such ranges are specifically recited herein so that amending any such subranges to explicitly recite them complies with requirements of description, sufficiency of description, and novelty of matter, including requirements under 35 USC § 112(a) and EPC Article 123(2). Unless expressly stated or otherwise required by context, all numerical parameters (representing values, ranges, amounts, percentages, etc.) set forth herein can also be read as if preceded by the word "about," even if the word "about" does not explicitly appear before the number. In addition, numerical parameters set forth herein should be construed in light of the numerical precision, that is, the number of reported significant digits, and by applying ordinary rounding techniques. It is also understood that the numerical parameters set forth herein necessarily have inherent variations characteristic of the underlying measuring methods used to determine the numerical value of the parameters. [Example]

[0535] The present invention will be more fully understood with reference to the following examples, which provide illustrative, non-limiting embodiments of the invention.

[0536] This example describes the use of the CRISPR system as an exemplary genome editing technology to create defined therapeutic genomic deletions, insertions, or replacements, collectively referred to herein as "genomic modifications," in the dystrophin gene (DMD gene), which result in the permanent deletion or correction of problematic exons from the genomic locus, restoring the dystrophin reading frame and restoring dystrophin protein activity.

[0537] Single-stranded gRNAs spanning various regions of the DMD gene were selected and tested for cleavage efficiency (Table 5). The gRNAs targeted exons, introns, and splice acceptors of multiple regions of interest in the DMD gene. The naming convention for all gRNAs discussed in the Examples is # (corresponding to the gRNA)-NN (Cas protein: SP - S. pyogenes, SA - S. aureus, NM - N. meningitides, ST - S. thermophiles, TD - T. denticola, Cpf1)-NN## (SA - splice acceptor, E - exon, I - intron). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0538] All tested gRNAs can be used for HDR / correction-based editing approaches. Single-stranded gRNAs targeting splice acceptors can be used to induce exon skipping to restore the reading frame of the DMD gene. Selected gRNA pairs can be used to create deletions in the DMD gene that restore the reading frame. Selected gRNA pairs can be used to create deletions that simulate patient mutations and generate model DMD mutant strains.

[0539] Various Cas orthologs were evaluated for cleavage. SP, NM, ST, SA, and Cpf1 gRNAs were delivered as RNA expressed from a U6 promoter in a plasmid or in a lentivirus. The corresponding Cas proteins were knocked into the cell line of interest for constitutive expression, delivered as mRNA, or delivered as protein. The activity of all gRNA formats listed above was assessed in HEK293T cells, K562 cells, or induced pluripotent stem cells (iPSCs) using TIDE analysis or next-generation sequencing.

[0540] Overall, it was determined that most gRNAs tested induced cleavage. However, the amount of cleavage was highly dependent on the Cas protein tested. In general, SP Cas9 gRNA was found to induce the highest level of cleavage, followed by SA Cas9 gRNA, which induced the second-highest level of cleavage. In general, it is beneficial to select gRNAs with the highest possible cleavage efficiency for therapeutic applications. However, for iPSC-based therapies, cleavage efficiency is less important. iPSCs are highly proliferative, simplifying the isolation of clonal populations of cells with the desired editing, even when editing efficiency is less than 10%.

[0541] The introduction of the defined therapeutic modifications described above represents a novel therapeutic strategy for the potential amelioration of DMD, as further described and exemplified herein. Example 1 CRISPR / SPCas9 target site in the dystrophin gene

[0542] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence NRG. The gRNA 20-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOS: 1-467,030. The gRNA 19-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOS: 1,410,430-1,410,472 in the Sequence Listing. Example 2 CRISPR / SACas9 target site in the dystrophin gene

[0543] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence NNGRRT. The gRNA 20-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOs: 467,031 to 528,196 in the Sequence Listing. Example 3 CRISPR / STCas9 target site in the dystrophin gene

[0544] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence NNAGAAW. The gRNA 24-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOs: 528,197 to 553,198 in the Sequence Listing. Example 4 CRISPR / TDCas9 target site in the dystrophin gene

[0545] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence NAAAAC. The gRNA 24-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOs: 553,199 to 563,911 in the Sequence Listing. Example 5 CRISPR / NMCas9 target site in the dystrophin gene

[0546] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence NNNNGHTT. The gRNA 24-bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOs: 563,912 to 627,854 and 1,410,400 to 1,410,402 in the Sequence Listing. Example 6 CRISPR / Cpf1 target site in the dystrophin gene

[0547] Regions of the dystrophin gene were scanned for target sites. Each region was scanned for a protospacer adjacent motif (PAM) with the sequence YTN. The gRNA 20-24 bp spacer sequences corresponding to the PAM were identified as shown in SEQ ID NOs: 627,855 to 1,410,399 and 1,410,403 to 1,410,429 in the Sequence Listing. Example 7 Exemplary genome editing strategies targeting exon 2

[0548] Some methods provide a gRNA pair that deletes exon 2 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 2 and the other gRNA cutting at the 3' end of exon 2. Example 8 Exemplary genome editing strategies targeting exon 8

[0549] Some methods provide a gRNA pair that deletes exon 8 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 8 and the other gRNA cutting at the 3' end of exon 8. Example 9 Exemplary genome editing strategies targeting exon 43

[0550] Some methods provide a gRNA pair that deletes exon 43 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 43 and the other gRNA cutting at the 3' end of exon 43. Example 10 Exemplary Genome Editing Methods Targeting Exon 44

[0551] Some methods provide a gRNA pair that deletes exon 44 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 44 and the other gRNA cutting at the 3' end of exon 44. Example 11 Exemplary genome editing strategies targeting exon 45

[0552] Some methods provide a gRNA pair that deletes exon 45 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 45 and the other gRNA cutting at the 3' end of exon 45. Example 12 Exemplary genome editing strategies targeting exon 46

[0553] Some methods provide a gRNA pair that deletes exon 46 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 46 and the other gRNA cutting at the 3' end of exon 46. Example 13 Exemplary genome editing strategies targeting exon 50

[0554] Some methods provide a gRNA pair that deletes exon 50 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 50 and the other gRNA cutting at the 3' end of exon 50. Example 14 Exemplary genome editing strategies targeting exon 51

[0555] Some methods provide a gRNA pair that deletes exon 51 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 51 and the other gRNA cutting at the 3' end of exon 51. Example 15 Exemplary genome editing strategies targeting exon 52

[0556] Some methods provide a gRNA pair that deletes exon 52 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 52 and the other gRNA cutting at the 3' end of exon 52. Example 16 Exemplary genome editing strategies targeting exon 53

[0557] Some methods provide a gRNA pair that deletes exon 53 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 53 and the other gRNA cutting at the 3' end of exon 53. Example 17 Exemplary genome editing strategies targeting exon 70

[0558] Some methods provide a gRNA pair that deletes exon 70 by cutting the gene twice, with one gRNA cutting at the 5' end of exon 70 and the other gRNA cutting at the 3' end of exon 70. Example 18 Exemplary genome editing strategies targeting exons 45-53

[0559] Some methods provide gRNA pairs that delete exons 45-53 by cutting the gene twice: one gRNA cuts at the 5' end of exon 45 and the other gRNA cuts at the 3' end of exon 53. Example 19 Exemplary genome editing strategies targeting exons 45-55

[0560] Some methods provide gRNA pairs that delete exons 45-55 by cutting the gene twice: one gRNA cuts at the 5' end of exon 45 and the other gRNA cuts at the 3' end of exon 55. Example 20 Bioinformatics analysis of the guide strand

[0561] Candidate guides are screened and selected through a multi-step process, including both theoretical binding and experimentally assessed activity.For example, in order to assess the possibility of effect at chromosomal locations other than the intended chromosomal location, as will be described and illustrated in more detail below, one or more of various bioinformatics tools that can be used to assess off-target binding can be used to assess candidate guides that have sequences that match specific on-target sites, such as the site in or near the dystrophin gene with adjacent PAM, for their potential to cut at off-target sites with similar sequences.Then, candidates that are predicted to have relatively low potential for off-target activity can be experimentally assessed to measure their on-target activity, and then the off-target activity at various sites.Preferred guides have sufficiently high on-target activity to achieve desired gene editing level at selected loci, and have relatively low off-target activity at other chromosomal loci to reduce the possibility of modification.The ratio of on-target to off-target activity is often referred to as the "specificity" of guides.

[0562] For initial screening of potential off-target activity, several known and publicly available bioinformatics tools can be used to predict the most likely off-target sites. Because binding to target sites in the CRISPR / Cas9 nuclease system is driven by Watson-Crick base pairing between complementary sequences, the degree of dissimilarity (and therefore the reduction in the potential for off-target binding) is essentially related to primary sequence differences, mismatches, and bulges (i.e., bases altered to non-complementary bases), as well as base insertions or deletions, at potential off-target sites relative to the target site. An exemplary bioinformatics tool called COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) (available online at crispr.bme.gatech.edu) aggregates such similarities. Other bioinformatics tools include, but are not limited to, GUIDO, autoCOSMID, and CCtop.

[0563] Bioinformatics was used to minimize off-target cleavage and reduce the deleterious effects of mutations and chromosomal rearrangements. Studies on the CRISPR / Cas9 system suggested the possibility of high off-target activity due to nonspecific hybridization of the guide strand to DNA sequences with base pair mismatches and / or bulges, especially at positions distal to the PAM region. Therefore, it is important to have bioinformatics tools that can identify potential off-target sites with insertions and / or deletions between the RNA guide strand and the genomic sequence in addition to base pair mismatches. A bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions), was developed. The COSMID (CRISPR 30D5 Target Sites and Deletions) was therefore used to search the genome for potential CRISPR 30D5 target sites (available online at crispr.bme.gatech.edu). The COSMID output ranks the list of potential off-target sites based on the number and position of mismatches, allowing for a more informed selection of target sites and avoiding the use of sites with more likely off-target cleavage.

[0564] We used an additional bioinformatics pipeline to compare the estimated on-target and / or off-target activity of gRNA targeting sites in a given region. Other features that can be used to predict activity include information about the cell type in question, DNA accessibility, chromatin state, transcription factor binding sites, transcription factor binding data, and other CHIP-seq data. We also compared additional factors that predict editing efficiency, such as the relative position and orientation of the gRNA pair, local sequence features, and microhomology. Example 21 Testing Preferred Guides in Cells for On-Target Activity

[0565] The gRNAs predicted to have the lowest off-target activity are then tested for on-target activity in human embryonic kidney-derived epithelial cells (HEK293T) by transient transfection and assessed for indel frequency using TIDE or next-generation sequencing. TIDE is a web tool for rapidly assessing CRISPR-Cas9 genome editing of target loci determined by guide RNAs (gRNAs or sgRNAs). Based on quantitative sequence trace data from two standard capillary sequencing reactions, the TIDE software quantifies editing efficacy and identifies the predominant types of insertions and deletions (indels) in the DNA of the target cell pool. For a detailed description and examples, see Brinkman et al., Nucl. Acids Res. (2014). Next-generation sequencing (NGS), also known as high-throughput sequencing, is an umbrella term used to describe several different modern sequencing technologies, including Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent:Proton / PGM sequencing, and SOLiD sequencing. These recent technologies allow DNA and RNA to be sequenced much faster and more inexpensively than the previously used Sanger sequencing, thus revolutionizing genomics and molecular biology research. HEK293T and iPSC cell types are known to have loose chromatin structures, making HEK293T an excellent model system for gene correction in iPSCs.

[0566] Chromatin is organized by coiling into discrete structures called nucleosomes. This coiling affects the accessibility of genomic material to the transcriptional machinery. Open genomic regions are called euchromatin, while tightly coiled regions are called heterochromatin. It is a well-accepted paradigm that stem cells generally have a loose chromatin conformation, and as cells differentiate into more specialized cell types, certain regions of the genome close to form heterochromatin (Sims, RJ, and D. Reinberg (2009), "Stem cells: Escaping fates with open states." Nature 460(7257):802-803). Example 22 Testing in relevant model cell lines

[0567] After all guide RNAs are individually evaluated and effective gRNAs are identified, all permutations of gRNA pairs are tested in relevant model cell lines for their ability to modify the DNA sequence of the dystrophin gene, which can be predicted to restore the dystrophin reading frame.Myoblasts and iPSC cell lines with modifications similar or identical to those found in patient samples are generated.If applicable, these cells are treated with various individual and pairwise combinations of gRNA and donor DNA template.The sample can then be evaluated for the restoration of dystrophin expression using one or more biological methods known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA), which specifically recognizes the C-terminus of dystrophin protein (note that truncated proteins do not contain an intact C-terminus).The gRNA pairs that restore dystrophin expression can then be further evaluated by additional biological techniques, such as Western blot, to confirm the expression of the appropriate size of dystrophin protein. Example 23 Testing different methods for HDR gene editing

[0568] After testing the gRNAs for both on-target and off-target activity, exon correction and knock-in strategies are tested for HDR gene editing.

[0569] For the exon correction approach, the donor DNA template is provided as a short single-stranded oligonucleotide, a short double-stranded oligonucleotide (PAM sequence intact / PAM sequence mutated), a long single-stranded DNA molecule (PAM sequence intact / PAM sequence mutated), or a long double-stranded DNA molecule (PAM sequence intact / PAM sequence mutated). In addition, the donor DNA template is delivered by AAV.

[0570] For DNA knock-in techniques, single- or double-stranded DNA having homology arms to the Xp21.2 locus can include 40 nt or more of the first targeted exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and the 3'UTR of the dystrophin gene, as well as at least 40 nt of the following intron. Single- or double-stranded DNA having homology arms to the Xp21.2 locus can include 80 nt or more of the first targeted exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and the 3'UTR of the dystrophin gene, as well as at least 80 nt of the following intron. The single- or double-stranded DNA having homology arms to the Xp21.2 locus may include 100 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and the 3'UTR of the dystrophin gene, and at least 100 nt of the following intron. The single- or double-stranded DNA having homology arms to the Xp21.2 locus may include 150 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and the 3'UTR of the dystrophin gene, and at least 150 nt of the following intron. The single-stranded or double-stranded DNA having arms of homology to the Xp21.2 locus may include 300 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete coding DNA sequence (CDS) of the dystrophin gene, and the 3'UTR of the dystrophin gene, as well as at least 300 nt of the following intron.The single-stranded or double-stranded DNA having homology arms to the Xp21.2 locus can include 400 nt or more of the first target exon (first coding exon) of the dystrophin gene, the complete CDS of the dystrophin gene, and the 3'UTR of the dystrophin gene, as well as at least 400 nt of the following intron. Alternatively, the DNA template is delivered by AAV.

[0571] For cDNA knock-in method, single-stranded or double-stranded cDNA can contain 40nt or more of the single exon target of dystrophin gene.Single-stranded or double-stranded cDNA can contain 80nt or more of the single exon target of dystrophin gene.Single-stranded or double-stranded cDNA can contain 100nt or more of the single exon target of dystrophin gene.Single-stranded or double-stranded cDNA can contain 150nt or more of the single exon target of dystrophin gene.Single-stranded or double-stranded cDNA can contain 300nt or more of the single exon target of dystrophin gene.Single-stranded or double-stranded cDNA can contain 400nt or more of the single exon target of dystrophin gene.Alternatively, DNA template is delivered by AAV.

[0572] For cDNA knock-in method, single-stranded or double-stranded cDNA can contain 40nt or more of multiple exon targets of dystrophin gene.Single-stranded or double-stranded cDNA can contain 80nt or more of multiple exon targets of dystrophin gene.Single-stranded or double-stranded cDNA can contain 100nt or more of multiple exon targets of dystrophin gene.Single-stranded or double-stranded cDNA can contain 150nt or more of multiple exon targets of dystrophin gene.Single-stranded or double-stranded cDNA can contain 300nt or more of multiple exon targets of dystrophin gene.Single-stranded or double-stranded cDNA can contain 400nt or more of multiple exon targets of dystrophin gene.Alternatively, DNA template is delivered by AAV. Example 24 Reassessment of lead CRISPR-Cas9 / DNA donor combinations

[0573] After testing various strategies for HDR gene editing, the lead CRISPR-Cas9 / DNA donor combination is reassessed in therapeutically relevant cells for deletion efficiency, recombination, and off-target specificity. Cas9 mRNA or RNP is formulated into lipid nanoparticles for delivery, sgRNA is formulated into nanoparticles or delivered as AAV, and donor DNA is formulated into nanoparticles or delivered as AAV. Example 25 In vivo testing in relevant animal models

[0574] After reassessing the CRISPR-Cas9 / DNA donor combination, lead formulations will be tested in vivo in therapeutically relevant mouse models.

[0575] Culturing in human cells allows for direct testing of the human target and background human genome, as explained above.

[0576] Preclinical efficacy and safety evaluation can be performed through engraftment of modified mouse or human cells in therapeutically relevant mouse models. Modified cells can be observed several months after engraftment. Example 26 Cleavage efficiency of S. pyogenes gRNAs targeting exons 45, 51, 53, 55, and 70 of the DMD gene

[0577] S. pyogenes (SP) gRNAs targeting exons 45, 51, 53, 55, and 70 of the DMD gene were tested (Figure 3A-3B). Each of exons 45, 51, 53, 55, and 70 may be edited using HDR / correction-based approaches.

[0578] The SP gRNA was cloned into a plasmid co-expressing the SP Cas protein. These plasmids were transfected into HEK293T cells using Lipofectamine 2000. Cells were harvested 48 hours post-transfection, genomic DNA was isolated, and cleavage efficiency was assessed using TIDE analysis. Data were aggregated from one experiment containing three to four replicates (N = 3-4). Data were plotted as mean and SEM.

[0579] The data from Figures 3A-3B show that most gRNAs cleave with greater than 50% efficiency in HEK293T cells. Example 27 Cleavage efficiency of gRNAs targeting the splice acceptors of exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the DMD gene

[0580] A viable option for treating DMD is to induce exon skipping to restore the reading frame of the DMD gene. To induce exon skipping, gene editing techniques must remove the AG sequence immediately upstream of the exon recognized by the endogenous splicing machinery. When a single-stranded gRNA induces a double-strand break, the cell repairs the break. During this time, the endogenous repair mechanism generates an error, inserting or deleting bases adjacent to the break site. A gRNA that mutates the AG sequence may induce exon skipping at this site because the splicing machinery can no longer recognize this site as a splice acceptor site and skips to the next splice acceptor in the adjacent exon.

[0581] S. pyogenes (SP), S. aureus (SA), S. thermophiles (ST), N. meningitides (NM), and Cpf1 gRNAs targeting the splice acceptor of exons 43, 44, 45, 46, 50, 51, 52, 53, and 55 of the DMD gene were designed and tested (Figures 4A, 4B, and 4C).

[0582] SP gRNA was designed to target the splice acceptor of exon 9 of the DMD gene. The gRNA was ordered as a split RNA gRNA from Integrated DNA Technologies (IDT). The split gRNA was annealed to tracRNA according to the manufacturer's instructions. The annealed split gRNA was then transfected into HEK293T cells stably expressing SP Cas9 protein using RNAiMax. Cells were harvested 48 hours post-transfection, genomic DNA was isolated, and cleavage efficiency was assessed using TIDE analysis (Figure 4A). Data were aggregated across two independent experiments, each containing three replicates (N = 2–6). Data were plotted as mean and SEM.

[0583] NM, ST, and SA gRNAs were designed to target the splice acceptors of nine exons. The gRNAs were clo...

Claims

**Claim 1** A composition comprising a guide RNA (gRNA) for use in the treatment of a patient with Duchenne muscular dystrophy, wherein the gRNA comprises a spacer sequence consisting of a) a sequence 19 to 25 nucleotides in length and b) an RNA version of the sequence set forth in SEQ ID NO: 1410450. **Claim 2** The composition according to claim 1, wherein the spacer sequence consists of the RNA version of SEQ ID NO: 1410450. **Claim 3** The composition according to any one of claims 1 to 2, wherein the gRNA is a single molecule gRNA (sgRNA) and / or the gRNA is a modified gRNA. **Claim 4** The composition according to any one of claims 1 to 2, wherein the gRNA further comprises a spacer extension sequence having a length greater than 1 nucleotide and less than 15 nucleotides. **Claim 5** The composition according to any one of claims 1, 2 or 4, wherein the gRNA further comprises a minimal CRISPR repeat sequence. **Claim 6** The composition according to any one of claims 1, 2, 4 or 5, wherein the gRNA further comprises a minimal tracrRNA sequence. **Claim 7** The composition according to any one of claims 1, 2 or 4 to 6, wherein the gRNA further comprises a 3'tracrRNA sequence. **Claim 8** The composition according to any one of claims 1, 2 or 4 to 7, wherein the gRNA further comprises a tracrRNA extension sequence. **Claim 9** The composition according to any one of claims 1 to 8, which is pre-complexed with a Cas9 endonuclease, optionally wherein the Cas9 endonuclease comprises a nuclear localization signal. **Claim 10** A composition comprising a nucleic acid for use in the treatment of a patient with Duchenne muscular dystrophy, wherein the nucleic acid encodes a gRNA according to any one of claims 1 to 8. **Claim 11** The composition according to claim 10, further comprising a second nucleic acid encoding a Cas9 endonuclease, optionally wherein the Cas9 endonuclease comprises a nuclear localization signal and optionally wherein the Cas9 endonuclease is the Cas9 endonuclease of Streptococcus pyogenes. **Claim 12** The composition for use according to any one of claims 10 or 11, which is delivered to cells by a viral vector. **Claim 13** The composition for use according to claim 12, wherein the viral vector is an adeno-associated virus (AAV) vector, and optionally the AAV vector is an AAV9 vector.

14. The composition for use according to any one of claims 1 to 13, wherein the treatment of a patient with Duchenne muscular dystrophy comprises editing the dystrophin gene in the cells of the patient.

15. The composition for use according to claim 14, wherein the cells are muscle cells or muscle progenitor cells.

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