Compositions and Methods for Treatment of DM1 with SLUCAS9 and SACAS9
AssigneeVertex Pharmaceuticals Incorporated
InventorsJohn Patrick Leonard, Zachary Michael Detwiler, Peter Thomas Hallock, Eric Gunnar Anderson
Life of this patent
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Abstract
Compositions and methods for treating myotonic dystrophy type 1 (DM1) with SluCas9 and SaCas9 are encompassed.
Claims177
A composition comprising:
- a. one or more guide RNAs (gRNAs), or a vector encoding one or more gRNAs, wherein each gRNA comprises:
- i. a spacer sequence selected from any one of SEQ ID NOs:
- 1-51, 53, 55-56, 58-59, 61-62, 64, 66, and 70;
- or
- ii. a spacer sequence that is at least 20 contiguous nucleotides of any one of SEQ ID NOs:
- 1-51, 53, 55-56, 58-59, 61-62, 64, 66, or 70;
- or
- iii. a spacer sequence that is at least 90% identical to any one of SEQ ID NOs:
- 1-51, 53, 55-56, 58-59, 61-62, 64, 66, or 70;
- wherein the gRNAs are for use with a SluCas9;
- and
- optionally a Staphylococcus lugdunensis Cas9 (SluCas9) or a nucleic acid encoding a SluCas9;
- or
- b. one or more guide RNAs (gRNAs), or a vector encoding one or more gRNAs, wherein each gRNA comprises:
- i. a spacer sequence selected from any one of SEQ ID NOs:
- 200-259;
- or
- ii. a spacer sequence that is at least 20 contiguous nucleotides of any one of SEQ ID NOs:
- 200-259;
- or
- iii. a spacer sequence that is at least 90% identical to any one of SEQ ID NOs:
- 200-259;
- wherein the gRNAs are for use with a Staphylococcus aureus Cas9 (SaCas9);
- and
- optionally a SaCas9 or a nucleic acid encoding a SaCas9.
A composition comprising:
- a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise:
- i. a first spacer sequence selected from SEQ ID NOs:
- 3, 5, 6, 9, 16, 21, 22, 25, 26, 30, 36, 38, 39, 40, 46, 51, 53, 55, 56, 58, 59, 61, 62, 64, 66, and 70, and a second spacer sequence selected from SEQ ID NOs:
- 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 27, 28, 29, 31, 32, 33, 34, 35, 37, 41, 42, 43, 44, 45, 47, 48, 49, and 50;
- and/or
- ii. a first spacer sequence having at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:
- 3, 5, 6, 9, 16, 21, 22, 25, 26, 30, 36, 38, 39, 40, 46, 51, 53, 55, 56, 58, 59, 61, 62, 64, 66, and 70, and a second spacer sequence having at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:
- 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 27, 28, 29, 31, 32, 33, 34, 35, 37, 41, 42, 43, 44, 45, 47, 48, 49, and 50;
- and/or
- iii. a first spacer 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:
- 3, 5, 6, 9, 16, 21, 22, 25, 26, 30, 36, 38, 39, 40, 46, 51, 53, 55, 56, 58, 59, 61, 62, 64, 66, and 70, and a second spacer 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:
- 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 27, 28, 29, 31, 32, 33, 34, 35, 37, 41, 42, 43, 44, 45, 47, 48, 49, and 50, wherein the gRNAs are for use with a SluCas9.
A composition comprising:
- a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise:
- i. a first spacer sequence selected from SEQ ID NOs:
- 201-203, 211, 215, 220, 225, 231, 235, 238, and 240-259 and a second spacer sequence selected from SEQ ID NOs:
- 200, 204-210, 212-214, 216-219, 221-224, 226-230, 232-234, 236-237, and 239;
- and/or
- ii. a first spacer sequence having at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:
- 201-203, 211, 215, 220, 225, 231, 235, 238, and 240-259, and a second spacer sequence having at least 20 contiguous nucleotides of a sequence selected from SEQ ID NOs:
- 1200, 204-210, 212-214, 216-219, 221-224, 226-230, 232-234, 236-237, and 239;
- and/or
- iii. a first spacer 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:
- 201-203, 211, 215, 220, 225, 231, 235, 238, and 240-259, and a second spacer 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:
- 200, 204-210, 212-214, 216-219, 221-224, 226-230, 232-234, 236-237, and 239, wherein the gRNAs are for use with a SaCas9.
A composition comprising:
- a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise:
- a) a first spacer sequence selected from SEQ ID NOs:
- 5, 21, 46, 55, 59, 61, or 64 and a second spacer sequence selected from SEQ ID NOs:
- 7, 19, 41, or 47, wherein the gRNAs are for use with a SluCas9;
- b) a first spacer sequence selected from SEQ ID NOs:
- 201-202 and a second spacer sequence selected from SEQ ID NOs:
- 206, 213, 218, or 224, wherein the gRNAs are for use with a SaCas9;
- c) a first and second spacer sequence of SEQ ID NOs:
- 5 and 7, wherein the gRNAs are for use with a SluCas9;
- d) a first and second spacer sequence of SEQ ID NOs:
- 5 and 10, wherein the gRNAs are for use with a SluCas9;
- e) a first and second spacer sequence of SEQ ID NOs:
- 5 and 19, wherein the gRNAs are for use with a SluCas9;
- f) a first and second spacer sequence of SEQ ID NOs:
- 5 and 41, wherein the gRNAs are for use with a SluCas9;
- g) a first and second spacer sequence of SEQ ID NOs:
- 5 and 47, wherein the gRNAs are for use with a SluCas9;
- h) a first and second spacer sequence of SEQ ID NOs:
- 21 and 7, wherein the gRNAs are for use with a SluCas9;
- i) a first and second spacer sequence of SEQ ID NOs:
- 21 and 19, wherein the gRNAs are for use with a SluCas9;
- j) a first and second spacer sequence of SEQ ID NOs:
- 21 and 41, wherein the gRNAs are for use with a SluCas9;
- k) a first and second spacer sequence of SEQ ID NOs:
- 21 and 47, wherein the gRNAs are for use with a SluCas9;
- l) a first and second spacer sequence of SEQ ID NOs:
- 46 and 7, wherein the gRNAs are for use with a SluCas9;
- m) a first and second spacer sequence of SEQ ID NOs:
- 46 and 10, wherein the gRNAs are for use with a SluCas9;
- n) a first and second spacer sequence of SEQ ID NOs:
- 46 and 19 wherein the gRNAs are for use with a SluCas9;
- o) a first and second spacer sequence of SEQ ID NOs:
- 46 and 41, wherein the gRNAs are for use with a SluCas9;
- p) a first and second spacer sequence of SEQ ID NOs:
- 46 and 47, wherein the gRNAs are for use with a SluCas9;
- q) a first and second spacer sequence of SEQ ID NOs:
- 55 and 7, wherein the gRNAs are for use with a SluCas9;
- r) a first and second spacer sequence of SEQ ID NOs:
- 55 and 19, wherein the gRNAs are for use with a SluCas9;
- s) a first and second spacer sequence of SEQ ID NOs:
- 55 and 41, wherein the gRNAs are for use with a SluCas9;
- t) a first and second spacer sequence of SEQ ID NOs:
- 55 and 47, wherein the gRNAs are for use with a SluCas9;
- u) a first and second spacer sequence of SEQ ID NOs:
- 59 and 7, wherein the gRNAs are for use with a SluCas9;
- v) a first and second spacer sequence of SEQ ID NOs:
- 59 and 19, wherein the gRNAs are for use with a SluCas9;
- w) a first and second spacer sequence of SEQ ID NOs:
- 59 and 41, wherein the gRNAs are for use with a SluCas9;
- x) a first and second spacer sequence of SEQ ID NOs:
- 59 and 47, wherein the gRNAs are for use with a SluCas9;
- y) a first and second spacer sequence of SEQ ID NOs:
- 61 and 7, wherein the gRNAs are for use with a SluCas9;
- z) a first and second spacer sequence of SEQ ID NOs:
- 61 and 10, wherein the gRNAs are for use with a SluCas9;
- aa) a first and second spacer sequence of SEQ ID NOs:
- 61 and 19, wherein the gRNAs are for use with a SluCas9;
- bb) a first and second spacer sequence of SEQ ID NOs:
- 61 and 41, wherein the gRNAs are for use with a SluCas9;
- cc) a first and second spacer sequence of SEQ ID NOs:
- 61 and 47, wherein the gRNAs are for use with a SluCas9;
- dd) a first and second spacer sequence of SEQ ID NOs:
- 64 and 7, wherein the gRNAs are for use with a SluCas9;
- ee) a first and second spacer sequence of SEQ ID NOs:
- 64 and 19, wherein the gRNAs are for use with a SluCas9;
- ff) a first and second spacer sequence of SEQ ID NOs:
- 64 and 41, wherein the gRNAs are for use with a SluCas9;
- gg) a first and second spacer sequence of SEQ ID NOs:
- 64 and 47, wherein the gRNAs are for use with a SluCas9;
- hh) a first and second spacer sequence of SEQ ID NOs:
- 202 and 218, wherein the gRNAs are for use with a SaCas9;
- ii) a first and second spacer sequence of SEQ ID NOs:
- 201 and 224, wherein the gRNAs are for use with a SaCas9;
- jj) a first and second spacer sequence of SEQ ID NOs:
- 202 and 213, wherein the gRNAs are for use with a SaCas9;
- or
- kk) a first and second spacer sequence of SEQ ID NOs:
- 202 and 206, wherein the gRNAs are for use with a SaCas9.
The composition of any one of the preceding claims, wherein the guide RNA comprises a crRNA and/or a tracrRNA sequence.
The composition of any one of the preceding claims, wherein the guide RNA is an sgRNA.
The composition of any one of the preceding claims, wherein the composition further comprises a pharmaceutically acceptable excipient.
The composition of any one of the preceding claims, wherein the guide RNA or nucleic acid encoding the guide RNA is associated with a lipid nanoparticle (LNP).
The composition of any one of the preceding claims, wherein the guide RNA or nucleic acid encoding the guide RNA is associated with a viral vector.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 1.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 2.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 3.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 4.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 5.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 6.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 7.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 8.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 9.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 10.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 11.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 12.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 13.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 14.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 15.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 16.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 17.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 18.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 19.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 20.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 21.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 22.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 23.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 24.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 25.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 26.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 27.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 28.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 29.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 30.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 31.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 32.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 33.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 34.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 35.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 36.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 37.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 38.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 39.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 40.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 41.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 42.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 43.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 44.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 45.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 46.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 47.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 48.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 49.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 50.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 51.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 53.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 55.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 56.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 58.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 59.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 61.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 62.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 64.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 66.
The composition of any one of the preceding claims comprising a spacer sequence having the sequence of SEQ ID NO:
- 70.
Use of a composition of any one of the preceding claims for the preparation of a medicament for treating a human subject having DM1.
Use of a composition of any one of the preceding claims for treating a human subject having DM1.
A method of excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene comprising delivering to a cell that comprises the TNR in the 3′ UTR of the DMPK gene a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise:
- i. a first spacer sequence selected from SEQ ID NOs:
- 3, 5, 6, 9, 16, 21, 22, 25, 26, 30, 36, 38, 39, 40, 46, 51, 53, 55, 56, 58, 59, 61, 62, 64, 66, and 70, and a second spacer sequence selected from SEQ ID NOs:
- 1, 2, 4, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 27, 28, 29, 31, 32, 33, 34, 35, 37, 41, 42, 43, 44, 45, 47, 48, 49, and 50, or a nucleic acid encoding the guide RNA;
- and
- ii. SluCas9 or a nucleic acid encoding the SluCas9, wherein at least one TNR is excised.
A method of treating a muscular dystrophy characterized by a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising delivering to a cell that comprises a TNR in the 3′ UTR of the DMPK gene:
- a. the composition of any one of 1b, 5, 6b, 6hh-6kk, 13-14, and 15-95;
- or
- b. a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise a first spacer sequence selected from SEQ ID NOs:
- 201-203, 211, 215, 220, 225, 231, 235, 238, and 240-259 and a second spacer sequence selected from SEQ ID NOs:
- 200, 204-210, 212-214, 216-219, 221-224, 226-230, 232-234, 236-237, and 239;
- g. a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprises SEQ ID NO:
- 201 and SEQ ID NO:
- 206;
- h. a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprises SEQ ID NO:
- 201 and SEQ ID NO:
- 224;
- i. a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprises SEQ ID NO:
- 202 and SEQ ID NO:
- 213;
- j. a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprises SEQ ID NO:
- 202 and SEQ ID NO:
- 218;
- and
- SluCas9 or a nucleic acid encoding the SaCas9.
A method of excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene comprising delivering to a cell that comprises the TNR in the 3′ UTR of the DMPK gene a pair of guide RNAs comprising a pair of spacer sequences, or one or more vectors encoding the pair of guide RNAs, wherein the pair of spacer sequences comprise:
- i. a first spacer sequence selected from SEQ ID NOs:
- 201-203, 211, 215, 220, 225, 231, 235, 238, and 240-259, and a second spacer sequence selected from SEQ ID NOs:
- 200, 204-210, 212-214, 216-219, 221-224, 226-230, 232-234, 236-237, and 239, or a nucleic acid encoding the guide RNA;
- and
- ii. SaCas9 or a nucleic acid encoding the SaCas9, wherein at least one TNR is excised.
Dependencies are inferred from the claim text.