US 12,281,327 B2
Lymphohematopoietic engineering using CAS9 base editors
Branden Moriarity, Minneapolis, MN (US); Beau Webber, Minneapolis, MN (US); Cara-Lin Lonetree, Minneapolis, MN (US); Miechaleen Diers, Minneapolis, MN (US); Mitchell Kluesner, Minneapolis, MN (US); Walker Lahr, Minneapolis, MN (US); and Emily Joy Pomeroy, Minneapolis, MN (US)
Assigned to REGENTS OF THE UNIVERSITY OF MINNESOTA, Minneapolis, MN (US)
Appl. No. 16/979,722
Filed by REGENTS OF THE UNIVERSITY OF MINNESOTA, Minneapolis, MN (US)
PCT Filed Mar. 13, 2019, PCT No. PCT/US2019/022049
§ 371(c)(1), (2) Date Sep. 10, 2020,
PCT Pub. No. WO2019/178225, PCT Pub. Date Sep. 19, 2019.
Claims priority of provisional application 62/642,151, filed on Mar. 13, 2018.
Prior Publication US 2021/0040507 A1, Feb. 11, 2021
Int. Cl. C12N 5/078 (2010.01); A61K 39/00 (2006.01); C12N 5/0783 (2010.01); C12N 15/90 (2006.01)
CPC C12N 15/907 (2013.01) [A61K 39/4611 (2023.05); A61K 39/4631 (2023.05); A61K 39/464412 (2023.05); C12N 5/0634 (2013.01); C12N 5/0636 (2013.01); C12N 2310/20 (2017.05); C12N 2510/00 (2013.01); C12N 2800/80 (2013.01)] 7 Claims
 
1. A method for producing a genetically engineered lymphohematopoietic cell, the method comprising
(a) introducing into a lymphohematopoietic cell:
(i) a plasmid, mRNA, or protein encoding a base editor fusion protein comprising a deaminase domain fused to a Cas9 nickase domain, wherein the nickase domain comprises a base excision repair inhibitor domain; and
(ii) three or more splice acceptor-splice donor (SA-SD) gRNAs having complementarity to a target nucleic acid sequence to be genetically modified, wherein the three or more SA-SD gRNAs each target a separate sequence selected from the group consisting of SEQ ID NOs: 1-15, and wherein sequences targeted include a PDCD1 gene, a TRAC gene, and a B2M gene; and
(b) culturing the introduced cell under conditions that promote disruption of splice sites targeted by the three or more SA-SD gRNAs, whereby the target nucleic acid sequence is modified by the base editor fusion protein and the three or more splice acceptor-splice donor (SA-SD) gRNAs relative to an untransfected lymphohematopoietic cell, and whereby a genetically engineered lymphohematopoietic cell is produced having reduced expression of TRAC, B2M, and PDCD1 gene products relative to an untransfected T cell.