CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING LEBER'S CONGENITAL AMAUROSIS 10 (LCA10)

• US 20150252358A1
• Filed: 03/10/2015
• Published: 09/10/2015
• Est. Priority Date: 03/10/2014
• Status: Active Grant

• Current Assignee: Editas Medicine, Inc.
• Original Assignee: Editas Medicine, Inc.
• Inventors: Bumcrot, David A., Maeder, Morgan L., Shen, Shen
• Granted Patent: US 9,938,521 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2750/14132 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 2750/14142 virus or viral particle as vehicle, e.g. encapsulating small organic molecule
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14145 Special targeting system for viral vectors
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

CRISPR/CAS-related compositions and methods for treatment of Leber'"'"'s Congenital Amaurosis 10 (LCA10) are disclosed.

Claims

1. A gRNA molecule comprising a targeting domain which is complementary with a target domain from the CEP290 gene.

2. The gRNA molecule of claim 1, wherein said targeting domain is configured to provide a cleavage event selected from a double strand break and a single strand break, within 10 nucleotides of a LCA10 target position.

3. The gRNA molecule of claim 1, wherein said targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, and Table 10.

50. The gRNA molecule of claim 1, wherein said gRNA is a modular gRNA molecule or a chimeric gRNA molecule.

52. The gRNA molecule of claim 1, wherein said targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 nucleotides or more in length.

246. A composition comprising the (a) a gRNA molecule of claim 1.

247. The composition of claim 246, further comprising (b) a Cas9 molecule and/or (c) a second gRNA molecule.

256. A method of altering a cell comprising contacting said cell with:

(a) a gRNA molecule of claim 1; and

(b) a Cas9 molecule.

257. The method of claim 256, comprising contacting said cell with (a), (b), and (c) a second gRNA molecule or a nucleic acid that encodes the second gRNA molecule.

258. The method of claim 256, wherein said cell is from a subject suffering from LCA10, or from a subject having a mutation at the LCA10 target position of the CEP290 gene.

261. The method of claim 256, wherein said contacting is performed ex vivo or in vivo.

272. A method of treating a subject, comprising contacting a subject or a cell from said subject with:

(a) a gRNA molecule of claim 1; and

(b) a Cas9 molecule.

273. The method of claim 272, further comprising contacting said subject with (a), (b), and (c) a second gRNA molecule.

274. The method of claim 272, wherein said subject is suffering from LCA10 or has a mutation at an LCA10 target position of the CEP290 gene.

279. The method of claim 272, wherein the contacting is performed ex vivo or in vivo.

297. A reaction mixture comprising:

(a) a gRNA molecule of claim 1, or a nucleic acid that encodes said gRNA molecule, and one or more of the following;

(b) a Cas9 molecule;

(c) a second gRNA molecule;

or

(d) a nucleic acid that encodes one or more of (b) and (c).

298. A kit comprising:

(a) gRNA molecule of claim 1, or a nucleic acid that encodes said gRNA molecule, and one or more of the following;

(b) a Cas9 molecule;

(c) a second gRNA molecule;

or

(d) a nucleic acid that encodes one or more of (b) and (c).

4-49. -49. (canceled)

51. (canceled)

53-68. -68. (canceled)

69. A nucleic acid that comprises:

(a) sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a LCA10 target domain in CEP290 gene.

72. The nucleic acid of claim 69, wherein said targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, and Table 10.

134. The nucleic acid of claim 69, further comprising:

(b) a sequence that encodes a Cas9 molecule and/or (c) a sequence that encodes a second gRNA molecule having a targeting domain that is complementary to a second target domain of the CEP290 gene.

136. The nucleic acid of claim 134, wherein said Cas9 molecule forms a double strand break or a single strand break in a target nucleic acid.

150. The nucleic acid of claim 134, wherein said targeting domain of said second gRNA molecule comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence from Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, and Table 10.

70-71. -71. (canceled)

73-133. -133. (canceled)

135. (canceled)

137-149. -149. (canceled)

151-215. -215. (canceled)

218-245. -245. (canceled)

248. (canceled)

249. A cell comprising a modification at the LCA10 target position of the CEP290 gene.

251. The cell of claim 249, wherein said cell comprises a gRNA molecule comprising a targeting domain that is complementary with a LCA10 target domain in the CEP290 gene.

252. The cell of claim 249, wherein said cell is a retinal cell, a photoreceptor cell, an induced pluripotent stem (iPS) cell, a cell derived from an iPS cell, a retinal progenitor cell, a cone photoreceptor cell or cone cell, a rod photoreceptor cell or rod cell, or a macular cone photoreceptor cell.

250. (canceled)

253-255. -255. (canceled)

259-260. -260. (canceled)

262-271. -271. (canceled)

275-278. -278. (canceled)

280-296. -296. (canceled)

299. (canceled)

300. A recombinant adenovirus-associated virus (AAV) genome comprising the following components:

306. The recombinant AAV genome of claim 300, wherein:

(a) the PII promoter is a CMV promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

401;

(b) the PII promoter is an EFS promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

402;

(c) the PII promoter is a GRK1 promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

403;

(d) wherein the PII promoter is a CRX promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

404;

(e) wherein the PII promoter is an NRL promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

405;

wherein the PII promoter is an RCVRN promoter, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

406.

324. The recombinant AAV genome of claim 300, further comprising a second gRNA component comprising a targeting domain and a scaffold domain,

wherein the targeting domain is 16-26 nucleotides in length and comprises, or consists of, a targeting domain sequence disclosed herein, in any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10;

and

wherein the scaffold domain comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO;

418;

337. The recombinant AAV genome of claim 300, comprising the nucleotide sequences of SEQ ID NOS:

408, 417, 418, 420, 421, 422, 424, and 437, and SEQ ID NO;

401, 402, 403, 404, 405, or 406;

or comprising a nucleotide sequence that is the same as, differs by no more than 100, 200, 300, 400, or 500 nucleotides, or has at least 90%, 92%, 94%, 96%, or 98% homology with, any of the nucleotide sequences of SEQ ID NOS;

428-433 or 445-450.

346. A recombinant AAV viral particle comprising the AAV genome of claim 300.

347. The recombinant AAV viral particle of claim 346 having any of the serotype in Table 24, or a combination thereof;

and/or having a tissue specificity of retinal pigment epithelium cells, photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells, or a combination thereof.

349. A method of producing the recombinant AAV viral particle, comprising providing the recombinant AAV genome of claim 300 and one or more capsid proteins under conditions that allow for assembly of an AAV viral particle.

350. A method of altering a cell comprising contacting the cell with the recombinant AAV viral particle of claim 346.

351. A method of treating a subject having or likely to develop LCA10 comprising contacting the subject or a cell from the subject with the recombinant viral particle of claim 346.

301-305. -305. (canceled)

307-323. -323. (canceled)

325-336. -336. (canceled)

338-345. -345. (canceled)

348. (canceled)

352-353. -353. (canceled)