Crispr-related methods and compositions

Patent metadata

Field	Value
Publication number	US-2016237455-A1
Application number	US-201415025222-A
Country	US
Kind code	A1
Filing date	Sep 26, 2014
Priority date	Sep 27, 2013
Publication date	Aug 18, 2016
Grant date	—

Abstract

Methods and compositions useful in targeting a payload to or editing a target nucleic acid utilizing CRISPR/Cas9 and guide RNA (gRNA) are disclosed herein

First claim

1 . A composition comprising a gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid from a gene or pathway implicated in a disease or disorder listed in Table VII IX-2.

2 - 124 . (canceled)

125 . The composition of claim 1 , comprising:
1a) one or more gRNA molecules; 1b) one or more Cas9 molecules; and 1c) optionally, a template nucleic acid; 2a) one or more gRNA molecules; 2b) one or more nucleic acids encoding one or more Cas9 molecules; and 2c) optionally, a template nucleic acid; 3a) one or more nucleic acids which encode one or more gRNA molecules; 3b) one or more Cas9 molecules; and 3c) optionally, a template nucleic acid; or 4a) one or more nucleic acids which encode one or more gRNA molecules; 4b) one or more nucleic acids encoding one or more Cas9 molecules; and 4c) optionally, a template nucleic acid.

126 - 153 . (canceled)

154 . A method of altering a target nucleic acid of a cell implicated in a disease or disorder listed in Table IX-2, comprising contacting said cell with
a composition comprising:
1a) one or more gRNA molecules;
1b) one or more Cas9 molecules; and
1c) optionally, a template nucleic acid;
2a) one or more gRNA molecules;
2b) one or more nucleic acids encoding one or more Cas9 molecules; and
2c) optionally, a template nucleic acid;
3a) one or more nucleic acids which encode one or more gRNA molecules;
3b) one or more Cas9 molecules; and
3c) optionally, a template nucleic acid; or
4a) one or more nucleic acids which encode one or more gRNA molecules;
4b) one or more nucleic acids encoding one or more Cas9 molecules; and
4c) optionally, a template nucleic acid.

155 - 180 . (canceled)

181 . A method of treating a subject by altering a target nucleic acid implicated in one or more of the diseases or disorders listed in Table IX-2, comprising administering to the subject, an effective amount of
a composition comprising:
1a) one or more gRNA molecules;
1b) one or more Cas9 molecules; and
1c) optionally, a template nucleic acid;
2a) one or more gRNA molecules;
2b) one or more nucleic acids encoding one or more Cas9 molecules; and
2c) optionally, a template nucleic acid;
3a) one or more nucleic acids which encode one or more gRNA molecules;
3b) one or more Cas9 molecules; and
3c) optionally, a template nucleic acid; or
4a) one or more nucleic acids which encode one or more gRNA molecules;
4b) one or more nucleic acids encoding one or more Cas9 molecules; and
4c) optionally, a template nucleic acid.

182 - 258 . (canceled)

259 . The composition of claim 125 , comprising one or more nucleic acids encoding a first gRNA molecule and a second gRNA molecule.

260 . The composition of claim 125 , comprising a first eaCas9 molecule and a second eaCas9 molecule.

261 . The composition of claim 125 , comprising one or more nucleic acids encoding a first eaCas9 molecule and a second eaCas9 molecule.

262 . The composition of claim 259 , wherein a first nucleic acid comprises a first promoter operably linked to a sequence encoding the first gRNA and a second nucleic acid comprises a second promoter operably linked to a sequence encoding the second gRNA.

263 . The composition of claim 261 , wherein a first nucleic acid comprises a first promoter operably linked to a sequence encoding the first eaCas9 and a second nucleic acid comprises a second promoter operably linked to a sequence encoding the second eaCas9.

264 . The composition of claim 181 , wherein one or both of the gRNA and the nucleic acid encoding the Cas9 has a modified ribophosphate backbone selected from phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.

265 . The composition of claim 264 , wherein a non-bridging phosphate oxygen atom in a phosphate backbone moiety in one or both of the gRNA and the nucleic acid encoding the Cas9 is replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R is hydrogen, alkyl, or aryl), C (an alkyl group, an aryl group), H, NR2 (wherein R is hydrogen, alkyl, or aryl), or OR (wherein R is alkyl or aryl).

266 . The composition of claim 264 , wherein one or both of the gRNA and the nucleic acid encoding the Cas9 has a modified ribophosphate backbone selected from morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

267 . The method of claim 181 , wherein the composition is delivered by a two-part delivery system, wherein the gRNA is delivered by a first delivery mode and the Cas9 is delivered by a second delivery mode.

268 . The method of claim 267 , wherein the two-part delivery results in reduced immunogenicity.

269 . The method of claim 154 , wherein the composition induces exon skipping.

270 . The method of claim 181 , wherein the composition induces exon skipping.

271 . The method of claim 154 , wherein the composition is delivered by a lipid nanoparticle (LNP).

272 . The method of claim 181 , wherein the composition is delivered by a lipid nanoparticle (LNP).

273 . The method of claim 154 , wherein the gRNA and the Cas9 protein are delivered as a complex.

274 . The method of claim 181 , wherein the gRNA and the Cas9 protein are delivered as a complex.

Assignees

• Editas Medicine Inc

Inventors

• Glucksmann Alexandra
• Palestrant Deborah
• Tartaglia Louis Anthony
• Mata-Fink Jordi
• Czechowicz Agnieszka Dorota

Classifications

• C12N15/85

  for animal cells · CPC title

• C12N15/8216

  Methods for controlling, regulating or enhancing expression of transgenes in plant cells · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title