IP Library › Granted Patent US 12,338,448
Granted Patent B2
US 12,338,448 · App. 16/086,127 · Granted Jun 24, 2025

Trans-replicating RNA

Inventors: Tim Beissert (Gross-Gerau, DE); Ugur Sahin (Mainz, DE); Mario Perkovic (Frankfurt, DE)
Assignees: TRON-Translationale Onkologie an der Universitatsmedizin der Johannes Gutenberg-Universitat Mainz; BIONTECH SE
C12N15/86C12N15/00C12N15/1131C12N15/64C12N15/79C12N2770/36141C12N2770/36143
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Quick Facts
Patent No.
US 12,338,448
App. No.
16/086,127
Granted
Jun 24, 2025
Kind
B2
Abstract

The present invention relates to systems and methods suitable for high-level protein production. In particular, a system comprising two separate RNA molecules is foreseen, each comprising a nucleotide sequence derived from an alphavirus: one RNA molecule comprises a RNA construct for expressing alphavirus replicase, and one RNA molecule comprises a RNA replicon that can be replicated by the replicase in trans. The system of the present invention enables expression of a protein of interest in a cell or organism, but is not associated with undesired virus-particle formation. The present invention is suitable for efficiently and safely producing a protein of interest in a target organism. Respective methods of protein production in vitro and in vivo as well as medical uses are provided herein. The present invention also provides DNA encoding the RNA molecules of the invention, and cells comprising the RNA molecules of the invention.

Claims (28)

1. A system comprising:

(a) an intron-free mRNA construct for expressing alphavirus replicase comprising

(i) a non-viral 5′ UTR,

(ii) an open reading frame encoding the replicase, and

(iii) a non-viral 3′ UTR, and

wherein the mRNA construct for expressing alphavirus replicase does not comprise an internal ribosomal entry site (IRES) element for driving translation of the replicase; and

(b) a RNA replicon that can be replicated by the replicase in trans,

wherein the mRNA construct of (a) for expressing alphavirus replicase comprises a 5′-cap for driving translation of the replicase.

2. The system according to claim 1 , wherein the 5′-cap is a natural 5′-cap or a 5′-cap analog.

3. The system according to claim 1 , wherein the open reading frame encoding the alphavirus replicase comprises the coding region(s) for non-structural proteins required for RNA replication.

4. The system according to claim 1 , wherein the mRNA construct for expressing alphavirus replicase comprises a 3′ poly(A) sequence.

5. The system according to claim 1 , wherein the mRNA construct for expressing alphavirus replicase cannot be replicated by the replicase.

6. The system according to claim 1 , wherein the RNA replicon comprises:

(1) an alphavirus 5′ replication recognition sequence, and

(2) an alphavirus 3′ replication recognition sequence.

7. The system according to claim 6 , wherein the alphavirus 5′ replication recognition sequence and the alphavirus 3′ replication recognition sequence direct replication of the RNA replicon in the presence of the replicase, and/or wherein the alphavirus 5′ replication recognition sequence and the alphavirus 3′ replication recognition sequence are native to the alphavirus from which the replicase is derived.

8. The system according to claim 1 , wherein the RNA replicon comprises a heterologous nucleic acid.

9. The system according to claim 1 , wherein the RNA replicon comprises an open reading frame encoding a protein of interest.

10. The system according to claim 9 , wherein the open reading frame encoding a protein of interest is non-native to the alphavirus from which the replicase is derived, and/or wherein expression of the open reading frame encoding a protein of interest is under the control of a subgenomic promoter.

11. The system according to claim 10 , wherein the subgenomic promoter is native to the alphavirus from which the replicase is derived, and/or wherein the subgenomic promoter is a promoter for a structural protein of an alphavirus.

12. The system according to claim 1 , wherein the RNA replicon comprises a 3′ poly(A) sequence, and/or a 5′-cap.

13. The system according to claim 1 , wherein the mRNA construct for expressing alphavirus replicase and/or the RNA replicon does not comprise an open reading frame encoding an intact alphavirus structural protein.

14. The system according to claim 1 , wherein the alphavirus is Semliki Forest virus or Venezuelan equine encephalitis virus or Sindbis virus or Chikungunya Virus.

15. The system according to claim 1 , wherein the mRNA is (+) strand mRNA.

16. The system according to claim 1 , wherein the mRNA is in vitro transcribed mRNA.

17. The system according to claim 16 , wherein the 5′-cap is a 5′-cap analog.

18. The system according to claim 1 , wherein the mRNA is not fully codon-optimized.

19. The system according to claim 1 , wherein the mRNA is comprised in a pharmaceutical composition comprising nanoparticles.

Assignments (4)
MERGER AND CHANGE OF NAME Recorded Sep 20, 2021
From: BIONTECH RNA PHARMACEUTICALS GMBH; BIONTECH SE
To: BIONTECH SE
Reel/Frame 057715/0788 →
CORRECTIVE ASSIGNMENT TO CORRECT THE ERROR IN THE NAME OF THE 2ND ASSIGNEE PREVIOUSLY RECORDED AT REEL: 047857 FRAME: 0050. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Nov 11, 2020
From: SAHIN, UGUR
To: TRON-TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITATSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAT MAINZ GGMBH; BIONTECH RNA PHARMACEUTICALS GMBH
Reel/Frame 054384/0683 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 27, 2018
From: SAHIN, UGUR
To: TRON-TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITATSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAT MAINZ GGMBH; BIOTECH RNA PHARMACEUTICALS GMBH
Reel/Frame 047857/0050 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 27, 2018
From: BEISSERT, TIM; PERKOVIC, MARIO
To: TRON-TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITATSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAT MAINZ GGMBH
Reel/Frame 047857/0156 →
Priority Claims (1)
WO PCT/EP2016/056160 · Mar 21, 2016 · international
Continuity (1)
Related Publication 20200299724A1 · Sep 24, 2020
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