Compositions and Methods Comprising a TTR Guide RNA and a Polynucleotide Encoding an RNA-Guided DNA Binding Agent

Abstract

Compositions and methods for editing, e.g., introducing double-stranded breaks, within the TTR gene are provided. Compositions and methods for treating subjects having amyloidosis associated with transthyretin (ATTR), are provided.

What is claimed is: 
     
         1 . A composition comprising:
 (i) a nucleic acid comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein:
 a. the open reading frame comprises a sequence with at least 93% identity to SEQ ID NO: 311; and/or 
 b. the open reading frame has at least 93% identity to SEQ ID NO: 311 over at least its first 50, 200, 250, or 300 nucleotides, or at least 95% identity to SEQ ID NO: 311 over at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides; and/or 
 c. the open reading frame consists of a set of codons of which at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the codons are codons listed in Table 4, the low A set of Table 5, or the low A/U set of Table 5; and/or 
 d. the open reading frame has an adenine content ranging from its minimum adenine content to 123% of the minimum adenine content; and/or 
 e. the open reading frame has an adenine dinucleotide content ranging from its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content; and 
   (ii) a guide RNA or a vector encoding a guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs: 5-72, 74-78, and 80-82.   
     
     
         2 . A method of modifying the TTR gene and/or inducing a double-stranded break (DSB) within the TTR gene, comprising delivering a composition to a cell, wherein the composition comprises:
 (i) a nucleic acid comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein:
 a. the open reading frame comprises a sequence with at least 93% identity to SEQ ID NO:311; and/or 
 b. the open reading frame has at least 93% identity to SEQ ID NO: 311 over at least its first 50, 200, 250, or 300 nucleotides, or at least 95% identity to SEQ ID NO: 311 over at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides; and/or 
 c. the open reading frame consists of a set of codons of which at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the codons are codons listed in Table 4, the low A set of Table 5, or the low A/U set of Table 5; and/or 
 d. the open reading frame has an adenine content ranging from its minimum adenine content to 123% of the minimum adenine content; and/or 
 e. the open reading frame has an adenine dinucleotide content ranging from its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content; and 
   (ii) a guide RNA or a vector encoding a guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs: 5-72, 74-78, and 80-82.   
     
     
         3 . A method of reducing TTR serum concentration, treating amyloidosis associated with TTR (ATTR), and/or reducing or preventing the accumulation of amyloids or amyloid fibrils comprising TTR in a subject, comprising administering a composition to a subject in need thereof, wherein the composition comprises:
 (i) a nucleic acid comprising an open reading frame encoding an RNA-guided DNA binding agent, wherein:
 a. the open reading frame comprises a sequence with at least 95% identity to SEQ ID NO:311; and/or 
 b. the open reading frame has at least 95% identity to SEQ ID NO: 311 over at least its first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides; and/or 
 c. the open reading frame consists of a set of codons of which at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the codons are codons listed in Table 4, the low A set of Table 5, or the low A/U set of Table 5; and/or 
 d. the open reading frame has an adenine content ranging from its minimum adenine content to 150% of the minimum adenine content; and/or 
 e. the open reading frame has an adenine dinucleotide content ranging from its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content; and 
   (ii) a guide RNA or a vector encoding a guide RNA, wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs: 5-72, 74-78, and 80-82, thereby reducing TTR serum concentration, treating amyloidosis associated with TTR (ATTR), and/or reducing or preventing the accumulation of amyloids or amyloid fibrils comprising TTR in the subject.   
     
     
         4 . The composition or method of any one of  claims 1 - 3 , wherein the guide RNA comprises a guide sequence selected from SEQ ID NOs: 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 22, 23, 27, 29, 30, 35, 36, 37, 38, 55, 61, 63, 65, 66, 68, or 69. 
     
     
         5 . The composition of  claim 1  or  4 , for use in inducing a double-stranded break (DSB) within the TTR gene in a cell or subject, modifying the TTR gene in a cell or subject, treating amyloidosis associated with TTR (ATTR) in a subject, reducing TTR serum concentration in a subject, or reducing or preventing the accumulation of amyloids or amyloid fibrils in a subject. 
     
     
         6 . The composition for use or method of any one of  claims 2 - 5 , wherein the method comprises administering the composition by infusion for more than 30 minutes, such as about 45-75 minutes, 75-105 minutes, 105-135 minutes, 135-165 minutes, 165-195 minutes, 195-225 minutes, 225-255 minutes, 255-285 minutes, 285-315 minutes, 315-345 minutes, 345-375 minutes, for about 1.5-6 hours, for about 60 minutes, about 90 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or about 240 minutes. 
     
     
         7 . The method or composition for use of any one of  claims 2 - 6 , wherein the composition reduces serum TTR levels, such as at least 50% as compared to serum TTR levels before administration of the composition, or by 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-98%, 98-99%, or 99-100% as compared to serum TTR levels before administration of the composition. 
     
     
         8 . The method or composition for use of any one of  claims 2 - 7 , wherein the composition results in editing of the TTR gene, optionally wherein the editing is calculated as a percentage of the population that is edited (percent editing), further optionally wherein the percent editing is between 30 and 99% of the population, such as between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the population. 
     
     
         9 . The method or the composition for use of any one of  claims 2 - 8 , wherein the composition reduces amyloid deposition in at least one tissue, optionally wherein the at least one tissue comprises one or more of stomach, colon, sciatic nerve, or dorsal root ganglion. 
     
     
         10 . The method or composition for use of  claim 9 , wherein amyloid deposition is measured 8 weeks after administration of the composition. 
     
     
         11 . The method or composition for use of any one of  claims 9 - 10 , wherein amyloid deposition is reduced by between 30 and 35%, 35 and 40%, 40 and 45%, 45 and 50%, 50 and 55%, 55 and 60%, 60 and 65%, 65 and 70%, 70 and 75%, 75 and 80%, 80 and 85%, 85 and 90%, 90 and 95%, or 95 and 99% of the amyloid deposition seen before administration of the composition. 
     
     
         12 . The method or composition for use of any one of  claims 2 - 11 , wherein the composition is administered or delivered at least two times. 
     
     
         13 . The method or composition for use of  claim 12 , wherein the administration or delivery occurs at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days or at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks or at an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months. 
     
     
         14 . The method or composition of any one of  claims 1 - 13 , wherein the guide RNA comprises a crRNA that comprises the guide sequence and further comprises a nucleotide sequence of SEQ ID NO: 126, wherein the nucleotides of SEQ ID NO: 126 follow the guide sequence at its 3′ end. 
     
     
         15 . The method or composition of any one of  claims 1 - 14 , wherein the guide RNA is a single guide (sgRNA). 
     
     
         16 . The method or composition of  claim 15 , wherein the sgRNA comprises a guide sequence that has the pattern of SEQ ID NO: 3. 
     
     
         17 . The method or composition of  claim 15  or  16 , wherein the sgRNA comprises the sequence of SEQ ID NO: 3. 
     
     
         18 . The method or composition of any one of  claims 15 - 17 , wherein the sgRNA comprises any one of the guide sequences of SEQ ID NOs: 5-72, 74-78, and 80-82 and the nucleotides of SEQ ID NO: 126. 
     
     
         19 . The method or composition of any one of  claims 15 - 17 , wherein the sgRNA comprises a sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID Nos: 87-113, 115-120, and 122-124. 
     
     
         20 . The method or composition of  claim 19 , wherein the sgRNA comprises a sequence selected from SEQ ID Nos: 87-113, 115-120, and 122-124. 
     
     
         21 . The method or composition of any one of  claims 1 - 20 , wherein the guide RNA comprises at least one modification. 
     
     
         22 . The method or composition of  claim 21 , wherein the at least one modification includes a 2′-O-methyl (2′-O-Me) modified nucleotide, a phosphorothioate (PS) bond between nucleotides, or a 2′-fluoro (2′-F) modified nucleotide. 
     
     
         23 . The method or composition of any one of  claims 21 - 22 , wherein the at least one modification includes a modification at one or more of the first five nucleotides at the 5′ end and/or one or more of the last five nucleotides at the 3′ end. 
     
     
         24 . The method or composition of any one of  claims 21 - 23 , wherein the at least one modification includes PS bonds between the first four nucleotides and/or between the last four nucleotides. 
     
     
         25 . The method or composition of any one of  claims 21 - 24 , wherein the at least one modification includes 2′-O-Me modified nucleotides at the first three nucleotides at the 5′ end and/or 2′-O-Me modified nucleotides at the last three nucleotides at the 3′ end. 
     
     
         26 . The method or composition of any one of  claims 21 - 25 , wherein the guide RNA comprises the modified nucleotides of SEQ ID NO: 3. 
     
     
         27 . The method or composition of any one of  claims 1 - 26 , wherein the guide RNA and the nucleic acid comprising an open reading frame encoding an RNA-guided DNA binding agent are associated with a lipid nanoparticle (LNP). 
     
     
         28 . The method or composition of  claim 27 , wherein the LNP comprises a CCD lipid, optionally wherein the CCD lipid is Lipid A or Lipid B, further optionally wherein the CCD lipid is lipid A. 
     
     
         29 . The method or composition of any one of  claims 27 - 28 , wherein the LNP comprises a helper lipid, optionally wherein the helper lipid is cholesterol. 
     
     
         30 . The method or composition of any one of  claims 27 - 29 , wherein the LNP comprises a stealth lipid (e.g., a PEG lipid), optionally wherein the stealth lipid is PEG2k-DMG. 
     
     
         31 . The method or composition of any one of  claims 27 - 30 , wherein:
 (i) the LNP comprises a lipid component and the lipid component comprises: about 50-60 mol-% amine lipid such as Lipid A, about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6;   (ii) the LNP comprises about 50-60 mol-% amine lipid such as Lipid A; about 27-39.5 mol-% helper lipid; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% stealth lipid (e.g., a PEG lipid), wherein the N/P ratio of the LNP composition is about 5-7 (e.g., about 6);   (iii) the LNP comprises a lipid component and the lipid component comprises: about 50-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10;   (iv) the LNP comprises a lipid component and the lipid component comprises: about 40-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6;   (v) the LNP comprises a lipid component and the lipid component comprises: about 50-60 mol-% amine lipid such as Lipid A; about 5-15 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 6;   (vi) the LNP comprises a lipid component and the lipid component comprises: about 40-60 mol-% amine lipid such as Lipid A; about 0-10 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10;   (vii) the LNP comprises a lipid component and the lipid component comprises: about 40-60 mol-% amine lipid such as Lipid A; less than about 1 mol-% neutral lipid; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-10;   (viii) the LNP comprises a lipid component and the lipid component comprises: about 40-60 mol-% amine lipid such as Lipid A; and about 1.5-10 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, wherein the N/P ratio of the LNP composition is about 3-10, and wherein the LNP composition is essentially free of or free of neutral phospholipid; or   (ix) the LNP comprises a lipid component and the lipid component comprises: about 50-60 mol-% amine lipid such as Lipid A; about 8-10 mol-% neutral lipid; and about 2.5-4 mol-% Stealth lipid (e.g., a PEG lipid), wherein the remainder of the lipid component is helper lipid, and wherein the N/P ratio of the LNP composition is about 3-7.   
     
     
         32 . The method or composition of any one of  claims 26 - 31 , wherein the LNP has an N/P ratio of about 6. 
     
     
         33 . The method or composition of  claim 26 - 32 , wherein the LNP comprises a neutral lipid, optionally wherein the neutral lipid is DSPC. 
     
     
         34 . The method or composition of any one of  claim 32 , wherein the LNP comprises a lipid component and the lipid component comprises: about 50 mol-% amine lipid such as Lipid A; about 9 mol-% neutral lipid such as DSPC; about 3 mol-% of stealth lipid such as a PEG lipid, such as PEG2k-DMG, and the remainder of the lipid component is helper lipid such as cholesterol wherein the N/P ratio of the LNP composition is about 6. 
     
     
         35 . The method or composition of any one of  claim 32 , wherein the LNP comprises a lipid component and the lipid component comprises: about 50 mol-% Lipid A; about 9 mol-% DSPC; about 3 mol-% of PEG2k-DMG, and the remainder of the lipid component is cholesterol wherein the N/P ratio of the LNP composition is about 6. 
     
     
         36 . The method or composition of any one of  claims 1 - 35 , wherein the RNA-guided DNA binding agent is a Cas cleavase. 
     
     
         37 . The method or composition of  claim 36 , wherein the RNA-guided DNA binding agent is Cas9. 
     
     
         38 . The method or composition of any one of  claims 1 - 37 , wherein the RNA-guided DNA binding agent is modified, optionally wherein the modified RNA-guided DNA binding agent comprises a nuclear localization signal (NLS). 
     
     
         39 . The method or composition of any one of  claims 1 - 38 , wherein the RNA-guided DNA binding agent is a Cas from a Type-II CRISPR/Cas system. 
     
     
         40 . The method or composition of any one of  claims 1 - 39 , wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier. 
     
     
         41 . The method or composition for use of  claim 40 , wherein non-homologous ending joining (NHEJ) leads to a mutation during repair of a DSB in the TTR gene that induces a frame shift or nonsense mutation in the TTR gene. 
     
     
         42 . The method or composition for use of  claim 41 , wherein a frame shift or nonsense mutation is induced in the TTR gene of at least 50% of liver cells. 
     
     
         43 . The method or composition for use of any one of  claims 41 - 42 , wherein a deletion or insertion of a nucleotide(s) occurs in the TTR gene at least 50-fold or more than in off-target sites. 
     
     
         44 . The method or composition of any one of  claims 1 - 43 , wherein the sequence of the guide RNA is:
 a) SEQ ID NO: 92 or 104;   b) SEQ ID NO: 87, 89, 96, or 113;   c) SEQ ID NO: 100, 102, 106, 111, or 112; or   d) SEQ ID NO: 88, 90, 91, 93, 94, 95, 97, 101, 103, 108, or 109,   
       optionally wherein the guide RNA does not produce indels at off-target site(s) that occur in a protein coding region in the genome of primary human hepatocytes. 
     
     
         45 . The method or composition for use of any one of  claims 2 - 44 , wherein administering the composition reduces levels of TTR in the subject. 
     
     
         46 . The method or composition for use of any one of  claims 2 - 45 , wherein the subject has ATTR, such as ATTRwt or hereditary ATTR. 
     
     
         47 . The method or composition for use of any one of  claims 2 - 46 , wherein the subject is human. 
     
     
         48 . The method or composition for use of any one of  claims 2 - 47 , wherein the subject has familial amyloid polyneuropathy, only or predominantly nerve symptoms of ATTR, familial amyloid cardiomyopathy, or only or predominantly cardiac symptoms of ATTR. 
     
     
         49 . The method or composition for use of any one of  claims 2 - 48 , wherein the subject expresses TTR having a V30 mutation, such as V30A, V30G, V30L, or V30M; the subject expresses TTR having a T60 mutation, such as T60A; the subject expresses TTR having a V122 mutation, such as V122A, V122I, or V122(−); or the subject expresses wild-type TTR. 
     
     
         50 . The method or composition for use of any one of  claims 2 - 49 , wherein after administration the subject has an improvement, stabilization, or slowing of change in symptoms of sensorimotor neuropathy, and/or the subject has an improvement, stabilization, or slowing of change in symptoms of congestive heart failure. 
     
     
         51 . The method or composition for use of any one of  claims 2 - 50 , wherein the composition or pharmaceutical formulation is administered via a viral vector. 
     
     
         52 . The method or composition for use of any one of  claims 2 - 50 , wherein the composition or pharmaceutical formulation is administered via lipid nanoparticles. 
     
     
         53 . The method or composition of any one of the preceding claims, wherein the sequence selected from SEQ ID NOs: 5-72, 74-78, and 80-82 is SEQ ID NO: 5, 6, 9, 13, 14, 15, 16, 17, 22, 23, 27, 30, 35, 36, 37, 38, 55, 63, 65, 66, 68, or 69. 
     
     
         54 . The composition or method of any one of  claims 1 - 53 , wherein the open reading frame comprises a sequence with at least 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 311. 
     
     
         55 . The composition or method of any one of  claims 1 - 54 , wherein at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the codons of the open reading frame are codons listed in Table 4, Table 5, or Table 7. 
     
     
         56 . The composition or method of any one of  claims 1 - 55 , wherein the open reading frame comprises a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 377. 
     
     
         57 . The composition or method of any of  claims 1 - 56 , wherein the nucleic acid is an mRNA in which at least 10% of the uridine is substituted with a modified uridine. 
     
     
         58 . The composition or method of  claim 57 , wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine. 
     
     
         59 . Use of a composition or formulation of any of  claims 1  or  4 - 58  for the preparation of a medicament for treating a human subject having ATTR.