Engineered post-poly A signal RNA and uses thereof

Patent metadata

Field	Value
Publication number	US-12203069-B2
Application number	US-202016875257-A
Country	US
Kind code	B2
Filing date	May 15, 2020
Priority date	May 17, 2019
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

The present disclosure is related to an engineered nucleic acid encoding a post-poly A signal RNA 3′ to a terminator for expression of protein, and/or non-coding RNA. Also provided herein are methods for reducing epigenetic silencing, genetic modification, transcriptional regulation of the engineered nucleic acid described herein.

First claim

What is claimed is:

1. An engineered nucleic acid comprising, from 5′ to 3′:
(1) a terminator, wherein the terminator comprises a nucleotide sequence encoding a poly A signal, and
(2) a nucleotide sequence encoding a post-poly A signal (post-PAS) RNA,
wherein the nucleotide sequence encoding the post-PAS RNA comprises a protein-coding cassette, wherein the protein-coding cassette from 5′ to 3′ comprises: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a 5′ cap, a nucleotide sequence encoding an internal ribosome entry site (IRES), a nucleotide sequence encoding the protein, a nucleotide sequence encoding a 3′ cap, and a nucleotide sequence encoding a second RNA cleavage site; or
wherein the nucleotide sequence encoding the post-PAS RNA comprises a non-coding RNA cassette,
(i) wherein the non-coding RNA cassette is a miRNA cassette that comprises from 5′ to 3′: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a primary micro-miRNA (pri-miRNA), and a nucleotide sequence encoding a second RNA cleavage site; or
(ii) wherein the non-coding RNA cassette is a guide RNA (gRNA) cassette that comprises from 5′ to 3′: a nucleotide sequence encoding a first RNA cleavage site, a nucleotide sequence encoding a gRNA, and a nucleotide sequence encoding a second RNA cleavage site.

2. The engineered nucleic acid of claim 1 , wherein the poly A signal comprises a nucleotide sequence of AAUAAA.

3. The engineered nucleic acid of claim 1 , wherein the terminator further comprises a poly A tail or a synthetic poly A mimic 3′ to the poly A signal.

4. The engineered nucleic acid of claim 3 , wherein the terminator comprises a nucleotide sequence encoding a RNA cleavage site between the poly A signal and the poly A tail or the synthetic poly A mimic, wherein the RNA cleavage site is capable of being cleaved by Cleavage and polyadenylation specificity factor (CPSF).

5. The engineered nucleic acid of claim 4 , wherein a nucleotide sequence (N) X is present between the poly A signal and the poly A tail or the synthetic poly A mimic, wherein N can be any nucleotide, and X is between 10 and 40, optionally wherein X is 25.

6. The engineered nucleic acid of claim 1 , wherein the nucleotide sequence encoding the post-PAS RNA comprises 1-10 protein-coding cassettes.

7. The engineered nucleic acid of claim 1 , wherein the nucleotide sequence encoding the post-PAS RNA comprises 1-10 miRNA cassettes.

8. The engineered nucleic acid of claim 1 , wherein the pri-miRNA further includes a miR-E element at the 3′ end of the pri-miRNA.

9. The engineered nucleic acid of claim 1 , wherein the nucleotide sequence encoding the post-PAS RNA comprises 1-10 gRNA cassettes.

10. The engineered nucleic acid of claim 1 , wherein the first and second RNA cleavage sites are nuclease sites.

11. The engineered nucleic acid of claim 1 , further comprising a promoter operably linked to a nucleic acid sequence encoding a transgene 5′ to the terminator.

12. A vector comprising the engineered nucleic acid of claim 1 .

13. An isolated cell comprising the engineered nucleic acid of claim 1 .

14. The engineered nucleic acid of claim 1 , wherein the first and second RNA cleavage sites are self-cleavage sites.

15. The engineered nucleic acid of claim 14 , wherein the self-cleavage sites are small self-cleaving ribozymes.

16. The engineered nucleic acid of claim 14 , wherein the small self-cleaving ribozymes, are selected from a hammerhead ribozyme, a hairpin ribozyme, a hepatitis delta virus (HDV) ribozyme, a Varkud satellite (VS) ribozyme, and a glmS ribozyme.

17. The engineered nucleic acid of claim 1 , wherein the first self-cleavage sites and the second self-cleavage sites are different self-cleavage sites.

Assignees

• Massachusetts Inst Technology

Inventors

• Weiss Ron
• Collins James J
• Vitu Elvira
• Enghuus Casper
• Gam Jeremy Jonathan

Classifications

• C12N2830/50

  regulating RNA stability, not being an intron, e.g. poly A signal · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N2506/03

  from non-embryonic pluripotent stem cells · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• C12N15/907

  in mammalian cells · CPC title