Methods and compositions for ocular cell therapy

Patent metadata

Field	Value
Publication number	US-2023348852-A1
Application number	US-202117997223-A
Country	US
Kind code	A1
Filing date	Apr 26, 2021
Priority date	Apr 27, 2020
Publication date	Nov 2, 2023
Grant date	—

Abstract

The present invention provides ocular cells, genetically modified by a CRISPR system targeting the expression of B2M for ocular cell therapy. The invention further provides methods of generating an expanded population of genetically modified ocular cells, for example limbal stem cells (LSCs) or corneal endothelial cells (CECs), wherein the cells are expanded involving the use of a LATS inhibitor and the expression of B2M in the cells has been reduced or eliminated. The present invention also provides cell populations, preparations, uses and methods of therapy comprising said cells.

First claim

1 . A modified limbal stem cell, which has reduced or eliminated expression of beta-2-microglobulin (B2M) relative to an unmodified limbal stem cell, wherein the B2M expression is reduced or eliminated by a CRISPR system comprising a gRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene.

2 . A modified limbal stem cell, which has reduced or eliminated expression of beta-2-microglobulin (B2M) relative to an unmodified limbal stem cell, wherein the B2M expression is reduced or eliminated by a CRISPR system comprising a nucleic acid molecule encoding a gRNA molecule comprising a targeting domain complementary to a target sequence in the B2M gene.

3 . The modified limbal stem cell of claim 1 or 2 , wherein the modified limbal stem cell was cultured in media comprising a large tumor suppressor kinase (“LATS”) inhibitor, optionally wherein the LATS inhibitor is a compound of Formula A1

or a salt thereof, wherein
X 1 and X 2 are each independently CH or N;
Ring A is
(a) a 5- or 6-membered monocyclic heteroaryl that is linked to the remainder of the molecule through a carbon ring member and comprises, as ring member, 1 to 4 heteroatoms that are independently selected from N, O and S, provided that at least one of the heteroatom ring member is an unsubstituted nitrogen (—N═) positioned at the 3- or the 4-position relative to the linking carbon ring member of the 5-membered heteroaryl or at the para ring position of the 6-membered heteroaryl; or
(b) a 9-membered fused bicyclic heteroaryl that is selected from

wherein “*” represents the point of attachment of ring A to the remainder of the molecule;
wherein ring A is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, cyano, C 1-6 alkyl, C 1-6 haloalkyl, —NH 2 , C 1-6 alkylamino, di-(C 1-6 alkyl)amino, C 3-6 cycloalkyl, and phenylsulfonyl;
R 0 is hydroxyl or C 1-6 alkoxy;
R 1 is hydrogen or C 1-6 alkyl;
R 2 is selected from
(a) C 1-8 alkyl that is unsubstituted or substituted by 1 to 3 substituents independently selected from
(i) halogen;
(ii) cyano;
(iii) oxo;
(iv) C 2 alkenyl;
(v) C 2 alkynyl;
(vi) C 1-6 haloalkyl;
(vii) —OR 6 , wherein R 6 is selected from hydrogen, C 1-6 alkyl that is unsubstituted or substituted by R 0 or —C(O)R 0 ;
(viii) —NR 7a R 7b , wherein R 7a is hydrogen or C 1-6 alkyl, and R 7b is selected from hydrogen, —C(O)R 0 , C 1-6 alkyl that is unsubstituted or substituted by —C(O)R 0 ;
(ix) —C(O)R 8 , wherein R 8 is R 0 or —NH—C 1-6 alkyl-C(O)R 0 ;
(x) -S(O) 2 C 1-6 alkyl;
(xi) monocyclic C 3-6 cycloalkyl or polycyclic C 7-10 cycloalkyl that are each unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, C 1- 6 alkyl, hydroxyC 1-6 alkyl, C 1-6 haloalkyl, R 0 , —NH 2 , C 1-6 alkylamino, and di-(C 1- 6 alkyl)amino;
(xii) 6-membered heterocycloalkyl comprising, as ring members, 1 to 2 heteroatoms independently selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from hydroxyl, halogen, C 1-6 alkyl, C 1-6 alkylamino, and di-(C 1-6 alkyl)amino;
(xiii) phenyl that is unsubstituted or substituted by halogen;
(xiv) 5- or 6-membered monocyclic heteroaryl comprising, as ring members, 1 to 4 heteroatoms independently selected from N and O; and
(xv) 9- or 10-membered fused bicyclic heteroaryl comprising, as ring member, 1 to 2 heteroatoms independently selected from N and O;

(b) -S(O) 2 C 1-6 alkyl;
(c) phenyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from halogen, C 1-6 alkyl and R 0 ;
(d) C 3-6 cycloalkyl that is unsubstituted or substituted by 1 to 2 substituents independently selected from C 1-6 haloalkyl, R 0 , C 1-6 alkylamino, di-(C 1-6 alkyl)amino, —C(O)R 0 , and C 1- 6 alkyl that is unsubstituted or substituted by R 0 or —C(O)R 0 ; and
(e) 4-membered heterocycloalkyl comprising, as ring members, 1 to 2 heteroatoms selected from N, O and S and that is unsubstituted or substituted by 1 to 2 substituents independently selected from C 1-6 haloalkyl, R 0 , C 1-6 alkylamino, di-(C 1- 6 alkyl)amino, —C(O)R 0 , and C 1-6 alkyl that is unsubstituted or substituted by R 0 or —C(O)R 0 ;

or R 1 and R 2 can be taken together with the nitrogen atom to which both are bound to form a 4- to 6-membered heterocycloalkyl that can include, as ring members, 1 to 2 additional heteroatoms independently selected from N, O, and S, wherein the 4- to 6-membered heterocycloalkyl formed by R 1 and R 2 taken together with the nitrogen atom to which both are bound is unsubstituted or substituted by 1 to 3 substituents independently selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, and R 0 ;
R 3 is selected from hydrogen, halogen and C 1-6 alkyl; and
R 5 is selected from hydrogen, halogen and —NH—(3- to 8-membered heteroalkyl), wherein the 3- to 8-membered heteroC 3-8 alkyl of the —NH—(3- to 8-membered heteroalkyl) comprises 1 to 2 oxygen atoms as chain members and is unsubstituted or substituted by R 0 .

4 . The modified limbal stem cell according to claim 3 , wherein the compound is selected from: dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine and N 1 ,N 1 ,3-trimethyl-N 3 -(2-(3-methyl-1H-pyrazol-4-yl)pyrido[3,4-d]pyrimidin-4-yl)butane-1,3-diamine.

5 . The modified limbal stem cell according to claim 3 , wherein the compound is dimethyl(3-methyl-3-{[2-(pyridin-4-yl)pyrido[3,4-d]pyrimidin-4-yl]amino}butyl)amine.

6 . The modified limbal stem cell according to any one of claims 3 to 5 , wherein the compound is present in a concentration of 3 to 10 micromolar.

7 . The modified limbal stem cell of claim any one of claims 1-6 , wherein the targeting domain of the gRNA molecule is complementary to a sequence within a genomic region selected from: chr15:44711469-44711494, chr15:44711472-44711497, chr15:44711483-44711508, chr15:44711486-44711511, chr15:44711487-44711512, chr15:44711512-44711537, chr15:44711513-44711538, chr15:44711534-44711559, chr15:44711568-44711593, chr15:44711573-44711598, chr15:44711576-44711601, chr15:44711466-44711491, chr15:44711522-44711547, chr15:44711544-44711569, chr15:44711559-44711584, chr15:44711565-44711590, chr15:44711599-44711624, chr15:44711611-44711636, chr15:44715412-44715437, chr15:44715440-44715465, chr15:44715473-44715498, chr15:44715474-44715499, chr15:44715515-44715540, chr15:44715535-44715560, chr15:44715562-44715587, chr15:44715567-44715592, chr15:44715672-44715697, chr15:44715673-44715698, chr15:44715674-44715699, chr15:44715410-44715435, chr15:44715411-44715436, chr15:44715419-44715444, chr15:44715430-44715455, chr15:44715457-44715482, chr15:44715483-44715508, chr15:44715511-44715536, chr15:44715515-44715540, chr15:44715629-44715654, chr15:44715630-44715655, chr15:44715631-44715656, chr15:44715632-44715657, chr15:44715653-44715678, chr15:44715657-44715682, chr15:44715666-44715691, chr15:44715685-44715710, chr15:44715686-44715711, chr15:44716326-44716351, chr15:44716329-44716354, chr15:44716313-44716338, chr15:44717599-44717624, chr15:44717604-44717629, chr15:44717681-44717706, chr15:44717682-44717707, chr15:44717702-44717727, chr15:44717764-44717789, chr15:44717776-44717801, chr15:44717786-44717811, chr15:44717789-44717814, chr15:44717790-44717815, chr15:44717794-44717819, chr15:44717805-44717830, chr15:44717808-44717833, chr15:44717809-44717834, chr15:44717810-44717835, chr15:44717846-44717871, chr15:44717945-44717970, chr15:44717946-44717971, chr15:44717947-44717972, chr15:44717948-44717973, chr15:44717973-44717998, chr15:44717981-44718006, chr15:44718056-44718081, chr15:4

Assignees

• Novartis Ag
• Intellia Therapeutics Inc

Inventors

• Berenshteyn Frada
• Han Bo
• Hao Xueshi
• Heyder Jessica
• Hoffman Timothy Z
• Jin Qihui
• Lacoste Arnaud
• Liu Jun
• Liu Yahu
• Mo Tingting
• Murray Bradley Andrew
• O’Connell Daniel Joseph
• Pan Jianfeng
• Xie Yun Feng
• Yan Shanshan
• Zou Yefen

Classifications

• C12N5/0621 Primary

  Eye cells, e.g. cornea, iris pigmented cells (photoreceptors C12N5/062) · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• C12N9/22

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

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title