RNA preparations comprising purified modified RNA for reprogramming cells
Inventors
Kariko, Katalin • Weissman, Drew • Dahl, Gary • Person, Anthony • Meis, Judith • Jendrisak, Jerome
Assignees
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Publication Number
US-12655395-B2
Publication Date
2026-06-16
Expiration Date
Abstract
The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.
Core Innovation
The invention relates to reprogramming a human or animal cell from a first differentiated state or phenotype to a second differentiated state or phenotype by introducing a purified RNA preparation comprising modified mRNA molecules that contain at least one modified nucleoside selected from pseudouridine and 5-methylcytosine and encode at least one reprogramming factor. The purified RNA preparation is substantially free of RNA contaminant molecules that activate an immune response or RNA sensors in somatic cells.
The disclosed contaminant categories include partial, smaller, larger, double-stranded, or un-capped mRNA molecules related to the reprogramming factor. The disclosure also includes variants where substantially all nucleosides are replaced with specified modified nucleosides and variants where all uridine nucleosides are replaced by pseudouridine nucleosides.
Named reprogramming factors include KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2. The second differentiated state can include a pluripotent state, with the disclosed possibility of transdifferentiation or redifferentiation.
Claims Coverage
The provided claim coverage centers on one independent reprogramming claim, with four additional inventive features expressed in dependent refinements.
Purified modified mRNA for phenotype reprogramming
Introducing into a human or animal cell that exhibits a first differentiated state a purified RNA preparation comprising modified mRNA molecules containing at least one modified nucleoside selected from pseudouridine and 5-methylcytosine and encoding at least one reprogramming factor, and culturing the cell under conditions wherein the cell exhibits a second differentiated state.
Immune-sensor avoidance by substantially contaminant-free purified RNA preparation
The purified RNA preparation is substantially free of RNA contaminants that activate RNA sensors in somatic cells.
Defined RNA contaminant categories
The RNA contaminant molecules are selected from partial, smaller, larger, double-stranded, or un-capped mRNA molecules related to the reprogramming factor.
Uridine-to-pseudouridine nucleoside replacement rule
All uridine nucleosides in the modified mRNA are replaced by pseudouridine nucleosides.
Selected reprogramming factors
The modified mRNA molecules encoding at least one reprogramming factor are selected from KLF4, LIN28, c-MYC, NANOG, OCT4, and SOX2.
The claims cover reprogramming human or animal cells using purified modified mRNA encoding reprogramming factors, with modified nucleosides and substantially free conditions for immune-activating RNA contaminants, together with defined contaminant categories, uridine-to-pseudouridine replacement, and specified reprogramming factors.
Stated Advantages
Reduces immune activation associated with RNA sensors in somatic cells and immune response activation.
Improves translation efficiency and RNA stability for nucleoside-modified RNA.
Promotes cell survival in culture and supports reprogramming outcomes.
Enables conversion of a first differentiated state or phenotype to a second differentiated state or phenotype, including pluripotent state, transdifferentiation, or redifferentiation.
Documented Applications
Reprogramming a human or animal cell from a first differentiated state or phenotype to a second differentiated state or phenotype using purified modified mRNA containing pseudouridine and/or 5-methylcytosine encoding at least one reprogramming factor.
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