US 11,672,874 B2
Methods and compositions for genomic integration
Daniel Getts, Stow, MA (US); Yuxiao Wang, Belmont, MA (US); Namita Bisaria, Somerville, MA (US); Inna Shcherbakova, Holliston, MA (US); and Socheata Ly, North Billerica, MA (US)
Assigned to MYELOID THERAPEUTICS, INC., Cambridge, MA (US)
Filed by Myeloid Therapeutics, Inc., Cambridge, MA (US)
Filed on Oct. 12, 2021, as Appl. No. 17/499,232.
Application 17/499,232 is a continuation in part of application No. PCT/US2020/049240, filed on Sep. 3, 2020.
Claims priority of provisional application 63/039,261, filed on Jun. 15, 2020.
Claims priority of provisional application 62/908,800, filed on Oct. 1, 2019.
Claims priority of provisional application 62/895,441, filed on Sep. 3, 2019.
Prior Publication US 2023/0067484 A1, Mar. 2, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); A61K 31/713 (2006.01); C12N 15/11 (2006.01); A61K 31/711 (2006.01); C07K 16/32 (2006.01); A61K 38/17 (2006.01); A61K 39/395 (2006.01); C07K 14/725 (2006.01); C12N 9/12 (2006.01); A61K 38/45 (2006.01); C12N 9/22 (2006.01); A61K 38/46 (2006.01); C12N 15/90 (2006.01); C12N 15/113 (2010.01); A61K 48/00 (2006.01)
CPC C12N 15/85 (2013.01) [A61K 31/711 (2013.01); A61K 31/713 (2013.01); A61K 38/1774 (2013.01); A61K 38/45 (2013.01); A61K 38/465 (2013.01); A61K 39/39558 (2013.01); C07K 14/7051 (2013.01); C07K 16/32 (2013.01); C12N 9/1276 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/113 (2013.01); C12N 15/907 (2013.01); C12Y 207/07049 (2013.01); A61K 48/00 (2013.01); C07K 2319/03 (2013.01); C07K 2319/09 (2013.01); C07K 2319/30 (2013.01); C07K 2319/33 (2013.01); C12N 2310/14 (2013.01); C12N 2310/20 (2017.05); C12N 2320/31 (2013.01); C12N 2800/80 (2013.01); C12N 2800/90 (2013.01); C12N 2830/50 (2013.01); C12N 2840/203 (2013.01)] 26 Claims
 
1. A method of expressing an exogenous human therapeutic polypeptide from a genomically integrated DNA sequence of a target human cell, the method comprising:
(a) contacting a composition to a population of human cells comprising the target human cell, the composition comprising one or more RNA molecules comprising a first RNA sequence and a second RNA sequence, wherein the ratio of the first RNA sequence to the second RNA sequence in the composition is at least 2:1, wherein the target human cell uptakes the one or more RNA molecules, and wherein:
(i) the first RNA sequence comprises a sequence that encodes a human ORF1p polypeptide, and
(ii) the second RNA sequence comprises (A) a sequence that encodes a human ORF2p polypeptide and (B) a sequence that is a reverse complement of a sequence encoding the exogenous human therapeutic polypeptide;
(b) translating the sequence of the first RNA sequence encoding the that encodes a human ORF1p polypeptide, thereby producing the human ORF1p polypeptide; and translating the sequence of the second RNA sequence encoding the human ORF2p polypeptide, thereby producing the human ORF2p polypeptide;
(c) reverse transcribing the sequence of (a)(ii)(B) via target-primed reverse transcription (TPRT) activity of the human ORF2p polypeptide translated in step (b), thereby producing a DNA sequence encoding the exogenous human therapeutic polypeptide;
(d) integrating the DNA sequence encoding the exogenous human therapeutic polypeptide produced in step (c) into genomic DNA of the target human cell; and
(e) expressing the exogenous human therapeutic polypeptide in the target human cell, wherein the exogenous human therapeutic polypeptide is expressed from the DNA sequence integrated into the genomic DNA of the target human cell in step (d).