Construct of self-circularization RNA

Inventors

LEE, SEONG-WOOK • Lee, Kyung Hyun • Han, Seung Ryul • Kim, Ji Hyun • KIM, SeongCheol

Assignees

Rznomics Inc

Abstract

A self-circularization RNA construct that can be expressed in a DNA vector and simultaneously circularized through a self-targeting and splicing reaction to form a circRNA is disclosed. The circRNA can consist only of a gene of interest which can be a coding, non-coding, or a combination thereof. The gene of interest has the advantage of being able to rapidly express a peptide or protein. The formed circRNA has a circular structure and has a stable and high half-life because 5′ and 3′ ends are not exposed. Accordingly, functional RNA such as miRNA, anti-miRNA, siRNA, shRNA, aptamer, functional RNA for gene/RNA editing, ADAR (adenosine deaminase acting on the RNA)-recruiting RNA, mRNA vaccine, mRNA therapeutic agent, vaccine adjuvant, and CAR-T mRNA can be produced as a stable circRNA in cells.

Core Innovation

A self-circularization RNA construct is described that comprises an internal guide sequence (IGS) region, a ribozyme region, a gene of interest (GOI) region, and a fragment containing a target splice site comprising uracil (U) at the 3′-end. The 5′-end guanine (G) of the IGS region and the 3′-end U of the target splice site-containing fragment form a G:U wobble base pair, and the IGS region comprises a sequence of 2 to 10 contiguous nucleotides that is reverse complementary to a 3′-end portion nucleotide sequence of the target splice site-containing fragment.

The ribozyme region is a Group I intron ribozyme, and the construct is configured to allow production of a circular RNA comprising the GOI region and the target splice site-containing fragment, while not including the ribozyme region and not including the IGS region. The described architecture directs self-circularization so that the circular RNA product retains the GOI and splice-site fragment features but excludes the ribozyme and IGS portions from the mature circular RNA.

Additional described embodiments refine the construct composition and the resulting circular RNA content using constraints and optional elements. These include narrowing the ribozyme region composition, defining what contiguous nucleotide sequences associated with the IGS and ribozyme form additional helix features while those specific sequences do not appear in the circular RNA, and including antisense-related sequence regions and additional structural sequence elements.

Claims Coverage

The independent claim defines the core construct architecture using an IGS region with a 5′-end G and defined reverse-complementarity, a Group I intron ribozyme region, a GOI region, and a 3′-end U in a target splice site-containing fragment that forms a G:U wobble base pair with the IGS G; the resulting circular RNA contains the GOI and target splice-site fragment but not the ribozyme or IGS. The remaining claim set includes dependent claims that refine these features by constraining sequence inclusion/exclusion, limiting component compositions, and specifying optional GOI sub-elements such as IRES and spacer, as well as antisense region length constraints.

The claim coverage is centered on a self-circularization RNA construct using a Group I intron ribozyme and a defined IGS/target splice site interaction to yield a circular RNA that contains the GOI and target splice site while excluding the ribozyme region and the IGS region. Dependent claims further narrow or refine component composition, inclusion/exclusion of specific nucleotide/helix-related sequences, and optional GOI sub-elements such as IRES and spacer.

Stated Advantages

Documented Applications