US 12,391,961 B2
Methods and products for transfecting cells
Matthew Angel, Cambridge, MA (US); and Christopher Rohde, Cambridge, MA (US)
Assigned to Factor Bioscience Inc., Cambridge, MA (US)
Filed by Factor Bioscience Inc., Cambridge, MA (US)
Filed on Jun. 9, 2023, as Appl. No. 18/332,621.
Application 18/332,621 is a continuation of application No. 16/913,306, filed on Jun. 26, 2020, granted, now 11,692,203.
Application 16/913,306 is a continuation of application No. 16/857,894, filed on Apr. 24, 2020, granted, now 10,829,738, issued on Nov. 10, 2020.
Application 16/857,894 is a continuation of application No. 16/776,765, filed on Jan. 30, 2020, granted, now 10,662,410, issued on May 26, 2020.
Application 16/776,765 is a continuation of application No. 16/567,059, filed on Sep. 11, 2019, granted, now 11,466,293, issued on Oct. 11, 2022.
Application 16/567,059 is a continuation of application No. 16/402,175, filed on May 2, 2019, granted, now 10,472,611, issued on Nov. 12, 2019.
Application 16/402,175 is a continuation of application No. 15/429,795, filed on Feb. 10, 2017, abandoned.
Application 15/429,795 is a continuation of application No. 15/222,453, filed on Jul. 28, 2016, granted, now 9,605,278, issued on Mar. 28, 2017.
Application 15/222,453 is a continuation of application No. 14/296,220, filed on Jun. 4, 2014, granted, now 9,422,577, issued on Aug. 23, 2016.
Application 14/296,220 is a continuation of application No. PCT/US2012/067966, filed on Dec. 5, 2012.
Claims priority of provisional application 61/664,494, filed on Jun. 26, 2012.
Claims priority of provisional application 61/637,570, filed on Apr. 24, 2012.
Claims priority of provisional application 61/569,595, filed on Dec. 12, 2011.
Claims priority of provisional application 61/566,948, filed on Dec. 5, 2011.
Prior Publication US 2023/0323399 A1, Oct. 12, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); A61K 35/28 (2015.01); C08K 5/5399 (2006.01); C12N 5/074 (2010.01); C12N 5/077 (2010.01); C12N 5/0789 (2010.01); C12N 9/16 (2006.01); C12N 9/22 (2006.01); C12N 15/87 (2006.01); C12N 15/90 (2006.01); C12P 21/00 (2006.01); H10F 19/80 (2025.01); A61K 35/12 (2015.01); C08G 77/08 (2006.01)
CPC C12N 15/87 (2013.01) [A61K 35/28 (2013.01); C08K 5/5399 (2013.01); C12N 5/0647 (2013.01); C12N 5/0657 (2013.01); C12N 5/0696 (2013.01); C12N 9/16 (2013.01); C12N 9/22 (2013.01); C12N 15/907 (2013.01); C12P 21/00 (2013.01); C12Y 301/21 (2013.01); H10F 19/80 (2025.01); A61K 2035/124 (2013.01); C08G 77/08 (2013.01); C12N 2500/25 (2013.01); C12N 2500/44 (2013.01); C12N 2501/115 (2013.01); C12N 2501/155 (2013.01); C12N 2501/165 (2013.01); C12N 2501/2303 (2013.01); C12N 2501/26 (2013.01); C12N 2501/91 (2013.01); C12N 2501/998 (2013.01); C12N 2506/09 (2013.01); C12N 2800/80 (2013.01); Y02E 10/50 (2013.01)] 20 Claims
 
1. A method for generating gene-edited cells, the method comprising:
(a) providing a plurality of cells comprising a target DNA sequence, wherein the plurality of cells is derived from a human subject;
(b) culturing the plurality of cells under conditions that allow the plurality of cells to proliferate; and
(c) contacting the plurality of cells in vitro or ex vivo with a plurality of synthetic RNA molecules,
wherein the plurality of synthetic RNA molecules comprises a nucleotide sequence that encodes a transcription activator-like effector nuclease,
wherein the plurality of synthetic RNA molecules is added to a medium surrounding the plurality of cells, and
wherein the contacting results in the plurality of cells internalizing the plurality of synthetic RNA molecules and expressing the transcription activator-like effector nuclease to result in a single-strand break or a double-strand break in the target DNA sequence, thereby generating the gene-edited cells.