US 12,203,069 B2
Engineered post-poly A signal RNA and uses thereof
Ron Weiss, Newton, MA (US); James J. Collins, Newton, MA (US); Elvira Vitu, Brookline, MA (US); Casper Enghuus, Somerville, MA (US); and Jeremy Jonathan Gam, Somerville, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on May 15, 2020, as Appl. No. 16/875,257.
Claims priority of provisional application 62/849,424, filed on May 17, 2019.
Prior Publication US 2020/0362345 A1, Nov. 19, 2020
Int. Cl. C07H 21/04 (2006.01); C12N 5/071 (2010.01); C12N 15/113 (2010.01); C12N 15/90 (2006.01)
CPC C12N 15/113 (2013.01) [C12N 5/0677 (2013.01); C12N 15/907 (2013.01); C12N 2310/141 (2013.01); C12N 2310/20 (2017.05); C12N 2506/03 (2013.01); C12N 2800/80 (2013.01); C12N 2830/50 (2013.01)] 17 Claims
 
1. An engineered nucleic acid comprising, from 5′ to 3′:
(1) a terminator, wherein the terminator comprises a nucleotide sequence encoding a poly A signal, and
(2) a nucleotide sequence encoding a post-poly A signal (post-PAS) RNA,
wherein the nucleotide sequence encoding the post-PAS RNA comprises a protein-coding cassette, wherein the protein-coding cassette from 5′ to 3′ comprises: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a 5′ cap, a nucleotide sequence encoding an internal ribosome entry site (IRES), a nucleotide sequence encoding the protein, a nucleotide sequence encoding a 3′ cap, and a nucleotide sequence encoding a second RNA cleavage site; or
wherein the nucleotide sequence encoding the post-PAS RNA comprises a non-coding RNA cassette,
(i) wherein the non-coding RNA cassette is a miRNA cassette that comprises from 5′ to 3′: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a primary micro-miRNA (pri-miRNA), and a nucleotide sequence encoding a second RNA cleavage site; or
(ii) wherein the non-coding RNA cassette is a guide RNA (gRNA) cassette that comprises from 5′ to 3′: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a gRNA, and a nucleotide sequence encoding a second RNA cleavage site.