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Circular RNA for translation in eukaryotic cells

A circular and nucleotide technology, applied in DNA/RNA fragments, recombinant DNA technology, nucleic acid vectors, etc., can solve problems such as short half-life

Pending Publication Date: 2021-02-23
MASSACHUSETTS INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a fundamental limitation of its use is its relatively short half-life in biological systems

Method used

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  • Circular RNA for translation in eukaryotic cells
  • Circular RNA for translation in eukaryotic cells
  • Circular RNA for translation in eukaryotic cells

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Experimental program
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Embodiment 1

[0179] There are three general strategies for exogenous RNA circularization: chemical methods using cyanogen bromide or similar condensing agents, enzymatic methods using RNA or DNA ligases, and ribozyme methods using self-splicing introns (Petkovic, S. &Muller, S., "RNA circularization strategies in vivo and in vitro," Nucleic Acids Research, 43(4):2454-2465 (2015); Beadudry, D. & Perreault, J., "An efficient strategy for the synthesis of circular RNA molecules," Nucleic Acids Research, 23(15):3064-3066 (1995); Micura, R., "Cyclic Oligoribonucleotides (RNA) by Solid-Phase Synthesis," Chemistry-A European Journal, 5(7):2077- 2082 (1999)). It has been reported that the ribozyme method using the substituted group I catalytic intron is more suitable for long RNA circularization, requiring only the addition of GTP and Mg2+ as cofactors (Petkovic, S. & Muller, S., "RNA circularization strategies in vivo and in vitro," Nucleic Acids Research, 43(4):2454-2465(2015)). This permuted ...

Embodiment 2

[0215] Circular RNAs (circRNAs) are a class of single-stranded RNAs with continuous structure, enhanced stability and lack of terminal motifs necessary for interaction with various cellular proteins. Here we show that unmodified exogenous circRNAs are able to bypass cellular RNA sensors and thus avoid immune responses in RIG-1 and toll-like receptor (TLR) competent cells and mice. It was found that the immunogenicity and protein expression stability of circRNA preparations depended on the purity, and a small amount of contaminating linear RNA would lead to a strong cellular immune response. Unmodified circRNAs were less immunogenic than unmodified linear mRNAs in vitro, partly due to evading TLR induction and eliciting a cytokine response similar to that induced by uridine-modified linear mRNAs. Furthermore, uridine modification of circRNAs was found to disrupt internal ribosome entry site (IRES)-mediated translation and had no significant effect on cytokine responses. Finall...

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Abstract

Disclosed are methods and constructs for engineering circular RNA. Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5' homology arm, b.) a 3' group I intron fragment containing a 3' splice site dinucleotide, c.) optionally, a 5' spacer sequence, d.) a protein coding or non-coding region, e.) optionally, a 3' spacer sequence, f.) a 5' Group I intron fragment containing a 5' splice site dinucleotide, and g.) a 3' homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In another embodiment, the vector can comprise the 5' spacer sequence, but not the 3' spacer sequence. In yet another embodiment,the vector can comprise the 3' spacer sequence, but not the 5' spacer sequence. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Application 62 / 851,548, filed May 22, 2019, U.S. Provisional Application 62 / 791,028, filed January 10, 2019, and U.S. Provisional Application 62 / 681,617, filed June 6, 2018 . The entire teachings of the aforementioned applications are incorporated herein by reference. [0003] The material in the ASCII text file is incorporated by reference. [0004] This application incorporates by reference the Sequence Listing contained in the following ASCII text file filed herewith: [0005] a) File name: 00502311003_FinalSequenceListing.txt; created on June 4, 2019, with a size of 55KB. [0006] governmental support [0007] This invention was made with government support under W32P4Q-13-1-0011 of the U.S. Department of Defense Advanced Research Projects Agency and 5R01HL125428 of the National Institutes of Health. The government has certain rights in this invention. Background technique [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088C12N15/115C12N15/64A61K31/7105
CPCA61K48/0025A61K48/005C12N2840/203C12N2840/44C12N2800/50C12N2830/42A61K31/7105C12N15/67C12N15/79Y02A50/30C12N15/85C12N2015/8518C12N2800/107C12N2999/007C07K16/2803C07K2317/31C12N2800/70C12N2800/202C12N2840/55C12N2840/60C12N2015/859C12N15/11A61K48/00
Inventor D·G·安德森R·A·韦塞尔赫夫特P·S·科瓦尔斯基
Owner MASSACHUSETTS INST OF TECH
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