RNA replicon for versatile and efficient gene expression
The present invention embraces a RNA replicon that can be replicated by a replicase of alphavirus origin. The RNA replicon comprises sequence elements required for replication by the replicase, but these sequence elements do not encode any protein or fragment thereof, such as an alphavirus non-structural protein or fragment thereof. Thus, in the RNA replicon according to the invention, sequence elements required for replication by the replicase and protein-coding region(s) are uncoupled. According to the present invention the uncoupling is achieved by the removal of at least one initiation codon compared to a native alphavirus genomic RNA. In particular, the RNA replicon comprises a 5′ replication recognition sequence, wherein the 5′ replication recognition sequence is characterized in that it comprises the removal of at least one initiation codon compared to a native alphavirus 5′ replication recognition sequence. The replicase of alphavirus origin may be encoded by an open reading frame on the RNA replicon or on a separate RNA molecule. The present invention enables efficient and safe expression of a protein of interest in a cell or organism, but is not associated with undesired production of fragments of alphavirus non-structural protein. Methods of protein production in vitro and in vivo, as well as medical uses, are provided herein.
1. A pharmaceutical composition comprising an RNA replicon and a pharmaceutically acceptable carrier or excipient,
wherein the RNA replicon comprises
(a) a 5′ replication recognition sequence which has at least 95% sequence identity to the sequence of SEQ ID NO: 5, wherein the 5′ replication recognition sequence is characterized in that it comprises the removal of all initiation codons compared to the native alphavirus 5′ replication recognition sequence as set forth in SEQ ID NO: 4, and
(b) a first open reading frame encoding a first transgene which is not derived from an alphavirus, wherein the 5′ replication recognition sequence and the open reading frame do not overlap and wherein the initiation codon of the open reading frame is the first initiation codon in the 5′ to 3′ direction of the RNA replicon.
2. The pharmaceutical composition according to claim 1 , wherein the 5′ replication recognition sequence comprises sequences homologous to conserved sequence element 1 and conserved sequence element 2 of an alphavirus.
3. The pharmaceutical composition according to claim 2 , wherein conserved sequence element 2 comprises a fragment of an open reading frame of a non-structural protein from an alphavirus.
4. The pharmaceutical composition according to claim 1 , which does not comprise an open reading frame encoding a truncated alphavirus non-structural protein; and/or which comprises a 3′ replication recognition sequence.
5. The pharmaceutical composition according to claim 1 , wherein the transgene encoded by the first open reading frame can be expressed from the RNA replicon as a template.
6. The pharmaceutical composition according to claim 1 , wherein the RNA replicon comprises a subgenomic promoter controlling production of subgenomic RNA comprising the first open reading frame encoding a transgene.
7. The pharmaceutical composition according to claim 6 , wherein the transgene encoded by the first open reading frame can be expressed from the RNA replicon and the subgenomic RNA.
8. The pharmaceutical composition according to claim 1 , wherein the RNA replicon comprises a subgenomic promoter controlling production of subgenomic RNA comprising a second open reading frame encoding a protein of interest.
9. The pharmaceutical composition according to claim 8 , wherein the subgenomic promoter and the second open reading frame encoding a protein of interest are located downstream from the first open reading frame encoding a transgene.
10. The pharmaceutical composition according to claim 8 , wherein the protein of interest encoded by the open reading frame is a second transgene or a functional alphavirus non-structural protein.
11. The pharmaceutical composition according to claim 10 , wherein the second open reading frame does not overlap with the 5′ replication recognition sequence; and/or wherein the RNA replicon can be replicated by the functional alphavirus non-structural protein.
12. The pharmaceutical composition according to claim 1 , wherein the RNA replicon does not comprise an open reading frame encoding functional alphavirus non-structural protein.
13. A pharmaceutical composition comprising a DNA, which DNA comprises a nucleic acid sequence encoding the RNA replicon of the pharmaceutical composition of claim 1 .