Materials and Methods for Treatment of Myotonic Dystrophy Type 1 (DM) and Other Related Disorders

Abstract

The present application provides materials and methods for treating a patient with one or more conditions associated with DMPK whether ex vivo or in vivo. In addition, the present application provides materials and methods for editing and/or modulating the expression of DMPK gene in a cell by genome editing.

1 - 53 . (canceled) 
     
     
         54 . A single-molecule guide RNA comprising: a spacer sequence that is an RNA sequence corresponding to any one of SEQ ID NOs: 5446, 5448, 5449, 5808, 5798, 5807, 5794, 5792, 5796, 5800, 5443, 5439, 5799, 5803, 5440, and 5444. 
     
     
         55 . The single-molecule guide RNA of  claim 54 , wherein the single-molecule guide RNA further comprises a spacer extension region. 
     
     
         56 . The single-molecule guide RNA of  claim 54 , wherein the single-molecule guide RNA further comprises a tracrRNA extension region. 
     
     
         57 . The single-molecule guide RNA of  claim 54 , wherein the single-molecule guide RNA is chemically modified. 
     
     
         58 . The single-molecule guide RNA of  claim 54  pre-complexed with a DNA endonuclease. 
     
     
         59 . The single-molecule guide RNA of  claim 58 , wherein the DNA endonuclease is a Cas9 or Cpfl endonuclease. 
     
     
         60 . The single-molecule guide RNA of  claim 59 , wherein the Cas9 or Cpfl endonuclease is selected from the group consisting of:  S.   pyogenes  Cas9,  S.   aureus  Cas9,  N.   meningitides  Cas9,  S.   thermophilus  CRISPR1 Cas9,  S.   thermophilus  CRISPR 3 Cas9,  T.   denticola  Cas9,  L.   bacterium  ND2006 Cpfl and  Acidaminococcus  sp. BV3L6 Cpfl, and variants having at least 90% homology to said endonucleases. 
     
     
         61 . The single-molecule guide RNA of  claim 60 , wherein the Cas9 or Cpfl endonuclease comprises one or more nuclear localization signals (NLSs). 
     
     
         62 . The single-molecule guide RNA of  claim 61 , wherein at least one of the one or more NLSs is at or within 50 amino acids of the amino-terminus of the Cas9 or Cpfl endonuclease and/or at least one of the one or more NLSs is at or within 50 amino acids of the carboxy-terminus of the Cas9 or Cpfl endonuclease. 
     
     
         63 . A DNA encoding the single-molecule guide RNA of  claim 54  . 
     
     
         64 . A method for editing a Dystrophia Myotonica-Protein Kinase (DMPK) gene in a cell by genome editing comprising: introducing into the cell one or more deoxyribonucleic acid (DNA) endonuclease or one or more polynucleotide encoding the one or more DNA endonuclease; and one or more single-molecule guide RNA comprising a spacer sequence that is an RNA sequence corresponding to any one of SEQ IDNOs: 5446, 5448, 5449, 5808, 5798, 5807, 5794, 5792, 5796, 5800, 5443, 5439, 5799, 5803, 5440, and 5444, to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the DMPK gene or DMPK regulatory elements that results in one or more permanent insertion, deletion, or mutation of at least one nucleotide within or near the DMPK gene, thereby reducing or eliminating the expression or function of aberrant DMPK gene products. 
     
     
         65 . The method of  claim 64 , wherein the cell is a muscle cell. 
     
     
         66 . The method of  claim 65 , wherein the muscle cell is a skeletal muscle cell, a smooth muscle cell, or a cardiac muscle cell. 
     
     
         67 . The method of  claim 64 , wherein the one or more polynucleotide encoding the one or more DNA endonuclease is codon optimized. 
     
     
         68 . The method of  claim 64 , wherein the one or more polynucleotide encoding the one or more DNA endonuclease is chemically modified. 
     
     
         69 . The method of  claim 64 , wherein the one or more DNA endonuclease is a Cas9 or Cpfl endonuclease. 
     
     
         70 . The method of  claim 64 , wherein the one or more DNA endonuclease is encoded in an AAV vector particle which also encodes the one or more single-molecule guide RNA. 
     
     
         71 . The method of  claim 64 , wherein the one or more DNA endonuclease is formulated in a liposome or lipid nanoparticle which also comprises the one or more single-molecule guide RNA. 
     
     
         72 . A kit comprising a single-molecule guide RNA comprising a spacer sequence that is an RNA sequence corresponding to any one of SEQ ID NOs: 5446, 5448, 5449, 5808, 5798, 5807, 5794, 5792, 5796, 5800, 5443, 5439, 5799, 5803, 5440, and 5444, or a polynucleotide encoding the single-molecule guide RNA. 
     
     
         73 . The kit of  claim 72 , further comprising:
 (a) a site-directed polypeptide;   (b) a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, adaptors for sequencing, or any combination thereof;   (c) instructions for using the components of the kit; or   (d) any combination of (a)-(c).