RNA replicon for versatile and efficient gene expression

Inventors

Beissert, Tim • Sahin, Ugur • Perkovic, Mario

Assignees

TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH • Biontech SE

Abstract

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.

Core Innovation

The invention relates to alphavirus RNA replicons used in cis-replication and trans-replication configurations for gene and/or protein expression. The RNA replicon comprises a 5′ replication recognition sequence having at least 95% sequence identity to a reference alphavirus sequence, and the 5′ replication recognition sequence comprises removal of all initiation codons compared to the native alphavirus 5′ replication recognition sequence.

A first open reading frame encoding a first transgene not derived from an alphavirus is provided so that the 5′ replication recognition sequence and the open reading frame do not overlap, and the initiation codon of the open reading frame is the first initiation codon in the 5′ to 3′ direction of the RNA replicon. In this architecture, replication recognition is maintained while translation from the 5′ replication recognition sequence is avoided.

The disclosure further describes embodiments in which the replicase is supplied in cis or in trans, with conserved sequence elements and stem-loop structures, optional 3′ replication recognition sequence, subgenomic promoter elements, and additional open reading frames to control production of subgenomic RNA transcripts and downstream expression. The replicon RNA also includes 5′ caps and cap analogs, 5′-UTR and 3′-UTR features, poly(A) sequence/poly(dA) cassette concepts, and constructs designed to avoid alphavirus structural proteins and to include no packaging signal.

Claims Coverage

The provided claims describe one independent claim directed to a pharmaceutical composition comprising an RNA replicon with three inventive features. The remaining claim material refines the defined replication recognition sequence, the non-overlapping transgene open reading frame, and the composition context.

Claim coverage centers on an RNA replicon in a pharmaceutical composition that preserves a defined 5′ replication recognition sequence with initiation codon removal and uses a non-overlapping first open reading frame encoding a non-alphavirus transgene that begins at the first initiation codon in the 5′ to 3′ direction.

Stated Advantages

Documented Applications