RNA replicon for reprogramming somatic cells
Inventors
Poleganov, Marco Alexander • Perkovic, Mario • Sahin, Ugur • Beissert, Tim
Assignees
TRON Translationale Onkologie an der Universitaetsmedizin der Johannes Gutenberg Universitaet Mainz gGmbH • Biontech SE
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Publication Number
US-12291717-B2
Publication Date
2025-05-06
Expiration Date
Abstract
The present invention embraces a RNA replicon that can be replicated by a replicase of alphavirus origin and comprises an open reading frame encoding a reprogramming factor. Such RNA replicons are useful for expressing a reprogramming factor in a cell, in particular a somatic cell. Cells engineered to express such reprogramming factors are useful in cell transplantation therapies.
Core Innovation
The invention relates to alphavirus-origin RNA replicon technology for reprogramming somatic cells to cells with stem cell characteristics, including pluripotent characteristics, for cell-therapy use. The system uses an RNA construct for expressing a functional alphavirus non-structural protein together with one or more RNA replicons that carry one or more open reading frames encoding reprogramming factors. The RNA construct is an mRNA molecule comprising a 5′-cap, and the RNA replicon is recognized by an alphaviral replicase in trans through a 5′ replication recognition sequence.
The RNA replicon is defined not to comprise an open reading frame encoding a functional alphavirus non-structural protein, and the 5′ replication recognition sequence is modified by removing initiation codons so that it does not overlap a first open reading frame and has the first functional initiation codon in the 5′→3′ direction. The described architectures include defined 5′ cap and UTR features, exclusion of structural protein and packaging signal content from the RNA replicon, and optional subgenomic promoter control. The systems further include one or multiple replicons encoding reprogramming factors such as OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28.
A further element described is the use of IFN-signaling inhibitors to enhance RNA expression viability in the context of RNA reprogramming toward embryonic-stem-like/iPS-cell characteristics. The patent text also discusses culture and characterization of dedifferentiated or reprogrammed cells as embryonic-stem-like, including marker expression, karyotypic analysis, and colony morphology.
Claims Coverage
The provided independent claims define four related aspects of the system: a two-RNA system, a set of RNA replicons encoding a set of reprogramming factors, a method of providing cells having stem cell characteristics, and cells comprising the system components. Across the independent claims, the inventive setup centers on alphavirus trans-replication with a specially constrained 5′ replication recognition sequence, coupled to stem-cell reprogramming factor expression while excluding an alphavirus non-structural protein open reading frame from the replicon.
Alphavirus trans-replication system with 5′ replication recognition sequence lacking initiation codons
An RNA replicon comprising an open reading frame encoding a reprogramming factor and a 5′ replication recognition sequence that can be recognized by an alphaviral replicase in trans and does not contain any initiation codons, does not overlap with a first open reading frame of the RNA replicon, and has the initiation codon of the first open reading frame as the first functional initiation codon in the 5′→3′ direction.
mRNA construct for expressing functional alphavirus non-structural protein with 5′-cap
The RNA construct is an mRNA molecule comprising a 5′-cap for expressing a functional alphavirus non-structural protein.
Replicon excludes alphavirus non-structural protein open reading frame
The RNA replicon does not comprise an open reading frame encoding a functional alphavirus non-structural protein.
Set of reprogramming-factor RNA replicons encoded with a reprogramming-factor set
A set of RNA replicons, wherein each RNA replicon comprises at least one open reading frame encoding a reprogramming factor and a 5′ replication recognition sequence with the same constraints, and wherein the set encodes a set of reprogramming factors.
Reprogramming method to provide cells having stem cell characteristics
Providing a cell population comprising somatic cells, providing an mRNA construct expressing a functional alphavirus non-structural protein, providing one or more RNA replicons configured to be replicated in trans and comprising open reading frames encoding reprogramming factors, introducing the RNA construct and the one or more RNA replicons into the somatic cells to express a set of reprogramming factors useful in reprogramming somatic cells to cells having stem cell characteristics, and allowing the development of cells having stem cell characteristics.
Cells comprising mRNA alphavirus non-structural protein construct plus reprogramming-factor replicon
Cells comprising an RNA construct for expressing a functional alphavirus non-structural protein and an RNA replicon comprising an open reading frame encoding a reprogramming factor and a 5′ replication recognition sequence characterized by being recognizable in trans, lacking initiation codons, not overlapping the first open reading frame, and having the first functional initiation codon in the 5′→3′ direction; with the RNA construct as an mRNA molecule comprising a 5′-cap and with the RNA replicon not comprising an open reading frame encoding a functional alphavirus non-structural protein.
Overall, the independent claims cover an alphavirus-derived RNA trans-replication design in which a capped mRNA expressing functional alphavirus non-structural proteins replicates, in trans, a replicon encoding reprogramming factor open reading frames via a 5′ replication recognition sequence constrained to avoid initiation codons and overlap, while ensuring the replicon itself does not encode functional alphavirus non-structural proteins. The scope is extended to systems with sets of reprogramming-factor replicons, to methods for producing cells having stem cell characteristics, and to cells containing the system components.
Stated Advantages
Modularity and separate preparation of replicase and replicon components are stated in the description as design features.
Reduced cost is stated via shorter replicase/reactive components as described in the patent text.
Avoids genome integration risks associated with prior iPS approaches, as characterized in the document.
Avoids prior-art trans-replicon AUG-driven nsP1/nsP4 fragment expression and related regulatory concerns by using a modified 5′ replication recognition sequence lacking initiation codons.
Uncouples replication signals from translation burden by preventing the engineered replicon from encoding functional alphavirus non-structural protein fragments.
Documented Applications
Reprogramming somatic cells to cells having stem cell characteristics, including embryonic-stem-like and iPS-cell colony characteristics, is described and characterized using stem-cell markers and colony morphology.
Providing differentiated cell types from stem-cell-characteristic cells by culturing under conditions that induce or direct partial or complete differentiation is described in the provided claims and summary context.
Cell therapy and the use of dedifferentiated or immature antigen presenting cells are mentioned in the provided description context.
Reprogramming somatic cells to cells with stem cell characteristics, including pluripotent characteristics, for cell-therapy use.
Differentiation of reprogrammed cells into multiple somatic lineages, including neuronal, hematopoietic, muscle, and epithelial lineages.
Potential autologous transplantation/cell therapy applications, including approaches described as avoiding embryo use.
Cell characterization using listed markers and endogenous genes including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E and endogenous OCT4, NANOG, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and TERT.
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