Self-replicating RNA and uses thereof
Inventors
MCGEE, Joshua Edward • KIRSCH, Jack Rainier • BRESSLER, Eric • Grinstaff, Mark W. • Wong, Wilson • Sertse, Lidya Yidnekachew • LI, Kexin
Assignees
Publication Number
US-12115217-B2
Publication Date
2024-10-15
Expiration Date
2043-11-17
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Abstract
The technology described herein is directed to compositions and methods for modifying and controlling the activity of cells by expression of proteins from self-amplifying RNA (saRNA). Also described herein are compositions and methods for modifying and controlling the activity of cells by expression of proteins from self-amplifying RNA that is substituted with chemically modified nucleotides.
Core Innovation
The invention is directed to compositions and methods for modifying and controlling the activity of cells by expression of proteins from self-amplifying RNA (saRNA), including those that are highly or fully substituted with chemically modified nucleotides. The saRNAs described are capable of undergoing self-replication in situ and are used to express genetically encodable proteins or non-coding RNAs in cells for therapeutic or research applications.
The background describes challenges in ex vivo cell engineering for adoptive cell therapy, such as cost, time, and risk of off-target genomic editing, as well as limitations of mRNA-based cell reprogramming due to insufficient expression level and duration, which often necessitate repeat dosing. The use of self-replicating RNA systems aims to address these limitations by enabling higher and prolonged expression of therapeutic proteins or gene programs and by providing mechanisms to control cell activity externally or via logic computation circuits.
A core innovation is the discovery that specific chemically modified nucleotides can be incorporated into saRNA at substitution levels greater than 25%, including up to and including 100%, without abrogating saRNA function. Contrary to previous teaching, these highly substituted saRNAs can maintain or even increase the level of cargo expression compared to unmodified saRNA, while also reducing innate immune signaling. The invention describes saRNA molecules comprising viral replication elements, subgenomic promoters, and one or more cargos of interest, with modifications that permit improved cellular delivery, controlled protein expression, and reduced immunogenicity.
Claims Coverage
There are three independent claims, each presenting a primary inventive feature: (1) highly substituted saRNA with specified modified nucleotides and cargos; (2) a defined structural format for the saRNA molecule; and (3) a method of expressing at least one cargo in a cell using the claimed saRNA.
Self-amplifying RNA comprising at least 25% modified nucleotides with specific functional groups and at least one cargo
The invention encompasses a self-amplifying RNA (saRNA) that includes at least 25% modified nucleotides. The modified nucleotides must be pyrimidine nucleoside phosphates with a moiety at carbon 5, chosen from methyl, ethyl, propyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, or hydroxypropyl functional groups. The saRNA further comprises at least one cargo of interest. The claim is not limited to one specific cargo and allows for single or multiple cargos as described.
Self-amplifying RNA with specified structural elements derived from viruses
The invention covers a self-amplifying RNA (saRNA) with a defined arrangement from 5′ to 3′ comprising: - a 5′ cap; - non-structural proteins nsp1, nsp2, nsp3, and/or nsp4 (each derived from at least one virus); - a subgenomic promoter (SGP) derived from at least one virus; - a 5′ untranslated region (UTR) derived from at least one virus; - at least one cargo of interest; - a 3′ untranslated region (UTR) derived from at least one virus; and - a poly-A tail. The structure supports the saRNA's replication and cargo expression functions and explicitly details the viral and genetic features necessary for self-amplification and expression.
Method of expressing at least one cargo in a cell by contacting with the claimed saRNA
The invention claims a method for expressing at least one cargo of interest in a cell. This is performed by contacting a cell with the patented self-amplifying RNA as defined above (including at least 25% modified nucleotides, with specified structural features and an encoded cargo). The claim encompasses the use of saRNA for intracellular delivery and expression of proteins or functional nucleic acids for modifying or controlling cell activity.
The patent claims cover highly and specifically modified self-amplifying RNAs, their detailed structural formats based on viral elements, and their application in expressing cargos in cells using the described saRNAs.
Stated Advantages
Enables incorporation of greater than 25%—up to 100%—of specified chemical modifications into saRNA without loss of function or expression of cargos.
Maintains or increases expression of cargos compared to unmodified saRNA, contrary to prior expectations in the field.
Reduces innate immune activation and early interferon response upon intracellular delivery, resulting in lower immunogenicity.
Enables higher and more durable protein or cargo expression than conventional mRNA or unmodified saRNA at equivalent or lower doses.
Permits robust and efficient cell modification ex vivo or in vivo, including hard-to-transfect cell types and primary immune cells.
Supports external and logic computation-based control of cell activity via encoded cargos responsive to inputs or circuit elements.
Documented Applications
Expression of chimeric antigen receptors (CARs) in immune cells for adoptive cell therapy, including externally controlled and logic-gated CAR systems.
Production of vaccines by expressing vaccine-associated antigens from viral, bacterial, or other pathogenic genomes for protective immunization.
Protein replacement therapies through expression of endogenous or therapeutic proteins, enzymes, growth factors, interleukins, or GLP-1.
Antibody therapies, including secretion of monoclonal antibodies, antibody fragments, or bispecific T cell engagers (BiTEs) from transfected cells.
Modification or reprogramming of various cell types ex vivo or in vivo, including human and animal cells, via direct RNA delivery (e.g., lipid nanoparticles, electroporation).
Transfection of cells for research or clinical applications using saRNA, enabling durable reporter gene expression and tracking.
Cell-, tissue-, or organ-specific gene expression, including applications requiring kidney-specific expression or cell-type restricted modification.
Therapeutic modulation of cellular differentiation, including induction of stem cell programs by saRNA-encoded transcription factors.
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