Materials and methods for treatment of friedreich ataxia and other related disorders

Abstract

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

1 .- 45 . (canceled) 
     
     
         46 . A single-molecule guide RNA comprising: at least a spacer sequence that is an RNA sequence corresponding to any one of SEQ ID NOs: 5911, 5916, 5917, 5923, 6625, 6627, 6651, 6664, 9174, 9193, 9194, 9195, 9201, 9203, 9204, 9206, 9207, 9211, 9212, 9214, 9224, 9227, 9228, 9232, 9233, 9234, 9237, 9241, 9248, 9250, 9251, 9252, 9253, 9255, 9257, 9258, 9260, 9264, 9268, 9271, 9272, 9282, 9283, 9289, 9293, 9311, 9321, 9327, 9328, 9329, 9333, 9345, 9346, 9353, 9356, 9359, 9360, 9361, 9363, 9364, 9366, 9369, 9372, 9373, 9374, 9377, 9378, 9379, 9380, 9387, 9388, 9513, 9516, 9521, 16064, 16072, 16081, 16084, 16091, 16094, 16096, 16100, 16109, 16112, 16120, 16122, 16123, 16128, 16129, 16131, 16132, 16133, 16134, 16137, 16138, 16141, 16143, 16144, 16155, 16157, 16161, 16163, 16164, 16175, 16176, 16177, 16178, 16185, 16187, 16201, 16221, 16224, 16238, 16240, 16242, 16245, 16246, 16252, 16253, 16254, 16256, 16257, 16259, 16260, 16263, 16271, 16273, 16274, 16275, 16276, 16278, 16290, 16294, 16295, 16307, 16311, 16315, 16450, 16451, 16453, 16454, 16457, 16459, and 37549. 
     
     
         47 . The single-molecule guide RNA of  claim 46 , wherein the single-molecule guide RNA further comprises a spacer extension region. 
     
     
         48 . The single-molecule guide RNA of  claim 46 , wherein the single-molecule guide RNA further comprises a tracrRNA extension region. 
     
     
         49 . The single-molecule guide RNA of claim  4 , wherein the single-molecule guide RNA is chemically modified. 
     
     
         50 . The single-molecule guide RNA of  claim 46 , wherein the single-molecule guide RNA is pre-complexed with one or more RNA-guided endonucleases, or one or more polynucleotides encoding the one or more RNA-guided endonucleases. 
     
     
         51 . The single-molecule guide RNA of  claim 50 , wherein the one or more RNA-guided endonucleases comprises a Cas9 or Cpf1 endonuclease. 
     
     
         52 . The single-molecule guide RNA of  claim 51 , wherein the Cas9 or Cpf1 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 Cpf1 and  Acidaminococcus  sp. BV3L6 Cpf1, and variants having at least 90% homology to said endonucleases. 
     
     
         53 . The single-molecule guide RNA of  claim 52 , wherein the Cas9 or Cpf1 endonuclease comprises one or more nuclear localization signals (NLSs). 
     
     
         54 . The single-molecule guide RNA of  claim 53 , wherein at least one NLS is at or within 50 amino acids of the amino-terminus of the Cas9 or Cpf1 endonuclease and/or at least one NLS is at or within 50 amino acids of the carboxy-terminus of the Cas9 or Cpf1 endonuclease. 
     
     
         55 . A DNA encoding the single-molecule guide RNA of  claim 46 . 
     
     
         56 . The single-molecule guide RNA of  claim 50 , wherein the one or more RNA-guided endonuclease is selected from any of those sequences in SEQ ID NOs: 1-620 and variants having at least 90% homology to any of the sequences set forth in SEQ ID NOs: 1-620. 
     
     
         57 . The single-molecule guide RNA of  claim 50 , wherein the single-molecule guide RNA is pre-complexed with one or more RNA encoding the one or more RNA-guided endonuclease. 
     
     
         58 . The single-molecule guide RNA of  claim 57 , wherein the one or more RNA is chemically modified. 
     
     
         59 . The single-molecule guide RNA of  claim 58 , wherein the one or more RNA is chemically modified in the coding region. 
     
     
         60 . The single-molecule guide RNA of  claim 57 , wherein the one or more RNA is codon optimized. 
     
     
         61 . The single-molecule guide RNA of  claim 46 , wherein the spacer sequence is an RNA sequence corresponding to any one of SEQ ID NOs: 16178, 16295, 16240, 9521, 16177, 9373, 9257, 16201, 16094, 9210, 9232, 16187, 9194, 16084, 16133, 16253, 16278, 9364, 16141, 9359, 16315, 9294, 16271, 16164, 16311, 9203, 9258, 16224, 16163, 16238, 16159, 9327, 9264, 16274, 9237, 9282, 16161, 16091, 16260, 16293, 16294, 16450, 16100, 16138, 9387, 9249, 16131, 9214, 37549, 16123, 16126, 9271, 16176, 9260, 16128, 9388, 16096, 16259, 9212, 9516, 9224, 9369, 9206, 9333, 16242, 9293, 16252, 9211, 16275, 9234, 16129, 16254, 9174, 9363, 9251, 16144, 9372, 16263, 16290, 16143, 16273, and 16245. 
     
     
         62 . The single-molecule guide RNA of  claim 46 , wherein the spacer sequence is an RNA sequence corresponding to any one of SEQ ID NOs: 5911, 5916, 5917, 5923, 6625, 6627, 6664, and 6651. 
     
     
         63 . The single-molecule guide RNA of  claim 49 , wherein the single-molecule guide RNA comprises one or more 2′—O-methyl phosphorothioate nucleotides. 
     
     
         64 . The single-molecule guide RNA of  claim 50 , wherein the pre-complexing comprises a covalent attachment of the single-molecule guide RNA to the one or more RNA-guided endonucleases. 
     
     
         65 . The single-molecule guide RNA of  claim 46 , wherein the single-molecule guide RNA is encoded in an AAV vector particle.