US 12,441,997 B2
Method of preparing self-circularized RNA
Fengmei Pi, Redmond, WA (US); Jarred Rensvold, Redmond, WA (US); and Cheng-Hsien Wu, Redmond, WA (US)
Assigned to GENSCRIPT USA INC., Redmond, WA (US)
Filed by GENSCRIPT USA INC., Redmond, WA (US)
Filed on Jul. 17, 2024, as Appl. No. 18/775,784.
Application 18/775,784 is a continuation of application No. PCT/US2023/084296, filed on Dec. 15, 2023.
Claims priority of provisional application 63/476,864, filed on Dec. 22, 2022.
Prior Publication US 2024/0368589 A1, Nov. 7, 2024
Int. Cl. C12N 15/113 (2010.01); C12N 15/11 (2006.01); C12N 15/66 (2006.01); C12N 15/85 (2006.01)
CPC C12N 15/111 (2013.01) [C12N 15/66 (2013.01); C12N 15/85 (2013.01); C12N 2310/532 (2013.01); C12N 2840/203 (2013.01)] 25 Claims
 
1. A method of preparing a circular ribonucleic acid (RNA), comprising:
transcribing a vector to form a precursor RNA comprising the following elements operably connected to each other and arranged in tandem in a 5′ to 3′ orientation:
a) a 5′ element,
b) a 3′ Group I self-splicing intron fragment,
c) none or an element comprising an internal ribosome entry site (IRES) and a protein coding region or an element comprising a noncoding region,
d) a 5′ Group I self-splicing intron fragment, and
e) a 3′ element;
wherein the 5′ element and the 3′ element form a thermodynamically stable multiple way junction RNA structure with a Gibbs free energy (ΔG) from −190 kcal/mol to −9.0 kcal/mol, provided that the stable structure is not a duplex with at least 95% base pairing between the 5′ element and the 3′ element,
wherein the 3′ Group I self-splicing intron fragment and the 5′ Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating the circular RNA,
wherein the thermodynamically stable multiple way junction RNA structure comprises a 4-way junction (4WJ) comprising
a first branch of the 4WJ domain is formed from a 5′ portion of a 4WJa sequence and a 3′ portion of a 4WJd sequence and comprises a first helical region,
a second branch of the 4WJ domain is formed from a 3′ portion of the 4WJa sequence and a 5′ portion of a 4WJb sequence and comprises a second helical region, and
a third branch of the 4WJ domain is formed from a 3′ portion of the 4WJb sequence and a 5′ portion of a 4WJc sequence and comprises a third helical region, and
a fourth branch of the 4WJ domain is formed from a 3′ portion of the 4WJc sequence and a 5′ portion of the 4WJd sequence and comprises a fourth helical region,
wherein each of said helical regions comprises a plurality of RNA nucleotide pairs that form canonical Watson-Crick bonds.