UTRs increasing the translation efficiency of RNA molecules
Inventors
Rudolph, Carsten • Aneja, Manish Kumar • Ferizi, Mehrije • GEIGER, Johannes
Assignees
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Publication Number
US-11136585-B2
Publication Date
2021-10-05
Expiration Date
Abstract
Described is an RNA molecule comprising (a) a coding region coding for a polypeptide; and (b) upstream of said coding region one or more UTR(s) comprising the sequence as shown in SEQ ID NO:1 or a sequence which shows 1 to 4 substitutions in comparison to SEQ ID NO:1 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:1; and/or (c) downstream of said coding region one or more UTR(s) comprising the sequence as shown in SEQ ID NO:2 or a sequence which shows 1 to 7 substitutions in comparison to SEQ ID NO:2 and which results in an RNA molecule having the same or a higher translation efficiency as an RNA molecule comprising an UTR comprising SEQ ID NO:2; wherein said polypeptide encoded by said coding region is not a cytochrome b-245 alpha polypeptide (CYBA). Moreover, described is a nucleic acid molecule encoding the RNA molecule according to the present invention. Further, described is a vector comprising the nucleic acid molecule according to the present invention and to a host cell comprising the vector according to the present invention. Further, described is a pharmaceutical composition comprising the RNA molecule according to the present invention and optionally a pharmaceutically acceptable carrier. Moreover, described is a kit comprising the RNA molecule according to the present invention. Finally, described is the use of one or more UTR(s) as defined in (b) and/or one or more UTR(s) as defined in (c) for increasing the efficiency of translating a coding region of an RNA molecule into a polypeptide or a protein encoded by said coding region.
Core Innovation
The document describes an RNA molecule comprising a coding region and one or more UTR(s) positioned upstream and/or downstream of the coding region. The upstream UTR(s) comprise the sequence shown in SEQ ID NO:1 or a sequence with 1 to 4 substitutions compared to SEQ ID NO:1 that results in the RNA molecule having the same or a higher translation efficiency than an RNA molecule comprising the UTR comprising SEQ ID NO:1.
The downstream UTR(s) comprise the sequence shown in SEQ ID NO:2 or a sequence with 1 to 7 substitutions compared to SEQ ID NO:2 that results in the RNA molecule having the same or a higher translation efficiency than an RNA molecule comprising the UTR comprising SEQ ID NO:2. The polypeptide encoded by the coding region is not a cytochrome b-245 alpha polypeptide (CYBA).
The work analyzes how combinations of upstream and downstream UTRs affect mRNA/protein expression kinetics, including predicted and observed effects on expression kinetics. It evaluates mRNA secondary structure, UTR end interaction, and an end-binding or circularization hypothesis, and reports higher and more sustained protein expression over time for UTR-stabilized reporter constructs in single-cell assays.
Claims Coverage
The independent claim covers an RNA molecule with specific upstream and/or downstream UTR sequences (SEQ ID NO:1 and SEQ ID NO:2) or limited-substitution variants that yield the same or higher translation efficiency, while excluding a CYBA-encoded polypeptide. Dependent claims further refine UTR placement and number, a minimum poly-A tail length, and broader formulation and method embodiments.
Utr sequence-defined RNA with same or higher translation efficiency
An RNA molecule comprising a coding region and one or more UTR(s) upstream of the coding region comprising the sequence as shown in SEQ ID NO:1 or a sequence with 1 to 4 substitutions that results in the RNA molecule having the same or a higher translation efficiency than an RNA molecule comprising an UTR comprising SEQ ID NO:1 and/or one or more UTR(s) downstream of the coding region comprising the sequence as shown in SEQ ID NO:2 or a sequence with 1 to 7 substitutions that results in the RNA molecule having the same or a higher translation efficiency than an RNA molecule comprising an UTR comprising SEQ ID NO:2.
Exclusion of CYBA-encoded polypeptide
The polypeptide encoded by the coding region is not a cytochrome b-245 alpha polypeptide (CYBA).
Specific 5′ and 3′ UTR placements
An RNA molecule wherein the upstream 5′-end UTR sequences comprise the sequence as shown in SEQ ID NO:1 and the downstream 3′-end UTR sequences comprise the sequence as shown in SEQ ID NO:2.
Asymmetric UTR number at coding region ends
An RNA molecule having one UTR at the 5′ end of the coding region and two UTRs at the 3′ end of the coding region.
Minimum poly-A tail length constraint
An RNA molecule having a poly-A tail with a length of at least 120 nucleotides.
RNA translation efficiency increasing method using defined UTRs
A method that increases the efficiency of translating an RNA coding region into its encoded polypeptide or protein by using one or more UTR(s) as defined in the claim language.
Formulation coverage for RNA-based compositions with optional carrier
A pharmaceutical composition including an RNA molecule as claimed, optionally with a pharmaceutically acceptable carrier, and inclusion of a nucleic acid molecule, a vector, or a host cell in the composition as further specified.
Overall, the claim set is centered on UTR-defined RNA molecules using SEQ ID NO:1 and SEQ ID NO:2, or limited-substitution variants, that achieve the same or higher translation efficiency, with explicit exclusion of CYBA as the encoded polypeptide. Dependent claims further specify UTR placement and number, a minimum poly-A tail length, broader delivery and formulation embodiments, and a method for increasing translation efficiency using the defined UTRs.
Stated Advantages
Same or higher translation efficiency for the RNA molecule compared with RNA molecules comprising the reference UTRs (SEQ ID NO:1 and SEQ ID NO:2).
Higher and more sustained protein expression over time for reporter mRNA constructs containing CYBA UTRs.
The method increases the efficiency of translating an RNA coding region into its encoded polypeptide or protein.
Documented Applications
RNA-based delivery/therapy embodiments for various diseases using coding regions encoding polypeptides other than CYBA, including nucleic acid, vectors, host cells, pharmaceutical compositions, and kits.
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