US 11,866,728 B2
Engineered retrons and methods of use
Alim Ladha, Cambridge, MA (US); Vladimir Presnyak, Cambridge, MA (US); Inna Shcherbakova, Cambridge, MA (US); Brian Goodman, Cambridge, MA (US); Mario Rodriguez Mestre, Cambridge, MA (US); Devin Scott Quinlan, Cambridge, MA (US); Muthusamy Jayaraman, Cambridge, MA (US); and Stephen Scully, Cambridge, MA (US)
Assigned to RENAGADE THERAPEUTICS MANAGEMENT INC., Cambridge, MA (US)
Filed by ReNAgade Therapeutics Management Inc., Cambridge, MA (US)
Filed on Dec. 22, 2022, as Appl. No. 18/087,673.
Claims priority of provisional application 63/373,545, filed on Aug. 25, 2022.
Claims priority of provisional application 63/370,880, filed on Aug. 9, 2022.
Claims priority of provisional application 63/301,936, filed on Jan. 21, 2022.
Prior Publication US 2023/0235365 A1, Jul. 27, 2023
Int. Cl. C12N 15/11 (2006.01); C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 9/12 (2006.01)
CPC C12N 15/907 (2013.01) [C12N 9/1276 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2310/531 (2013.01); C12N 2800/80 (2013.01)] 30 Claims
 
1. A gene editing system comprising one or more delivery vehicles, wherein:
the delivery vehicle(s) comprise RNA cargo,
said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the nucleic acid programmable nuclease;
wherein the engineered retron ncRNA comprises an HDR nucleotide sequence substituted into a retron ncRNA;
wherein the retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to any one of SEQ ID NO:15833, SEQ ID NO:18547, or SEQ ID NO:19053;
wherein the HDR nucleotide sequence is substituted at a hairpin loop of the retron ncRNA;
wherein the retron reverse transcriptase and the retron ncRNA are from the same phylogenetic clade;
each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c),
whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c).