Alternative nucleic acid molecules containing reduced uracil content and uses thereof

Inventors

Hoge, Stephen G. • ISSA, William Joseph • Miracco, Edward John • Nelson, Jennifer • Reynders, John • Stanton, Matthew

Assignees

ModernaTx Inc • Moderna Inc

Abstract

The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.

Core Innovation

The invention relates to engineering polynucleotides by incorporating microRNA binding sites to achieve cell-type- and tissue-specific expression regulation. It describes microRNA binding sites within polynucleotides such as mRNA and 3′ UTR and 5′ UTR contexts, including tissue-enriched miRNAs. The binding sites include nervous system, pancreas, kidney, muscle, endothelial, epithelial, and embryonic stem cell–enriched miRNAs.

The invention further addresses immune evasion by incorporating miRNA binding sites intended to dampen antigen presentation by antigen-presenting cells (APCs). It includes approaches for tuning miRNA effects, including using seed sequence identity and seed mutations, and introducing miRNA motifs into structured RNA elements, including stem-loop-containing elements, to regulate miRNA-mediated activity.

In additional implementations, RNA-binding protein (RBP) motif and RNA-binding domain (RBD) incorporation is described in connection with engineered structured RNA elements, to affect stability and translation control. The document also relates to an mRNA encoding a polypeptide, where the mRNA includes a 5′-cap structure, a 5′-UTR, an open reading frame (ORF), a 3′-UTR, and a poly-A region, and the ORF includes at least 95% 5-methoxyuracils with uracil content between the theoretical minimum and 150% of the theoretical minimum.

The mRNA further provides increased expression in a mammalian cell of the encoded polypeptide relative to a reference mRNA. The invention also provides a method of producing a modified mRNA by replacing at least one codon containing a uracil with a codon encoding the same amino acid but containing a lower number of uracils, and a composition that includes a lipid nanoparticle comprising a cationic lipid together with the mRNA, optionally with a 3′-UTR containing at least one microRNA (miRNA) binding site.

Claims Coverage

The independent claims cover seven core areas: engineered polynucleotides with microRNA binding sites, tuning miRNA effects with seed sequence identity or seed mutations, structured RNA elements with RNA-binding protein motifs and RNA-binding domains, an mRNA construct, a method for producing modified mRNA, and a lipid nanoparticle composition containing the mRNA. Across the claims, the coverage centers on cell-type- and tissue-specific expression regulation, antigen presentation by antigen-presenting cells (APCs), stability and translation control, and ORF uracil substitution with 5-methoxyuracil and specified bounds on ORF uracil content relative to a theoretical minimum, together with increased expression in a mammalian cell relative to a reference mRNA.

Across the independent claims, the primary inventive coverage centers on engineered polynucleotides with microRNA binding sites, structured RNA elements with RNA-binding protein motifs and RNA-binding domains, and mRNAs, a production method, and a lipid nanoparticle composition that require at least 95% of uracils to be 5-methoxyuracils and impose a specific uracil-content range relative to a theoretical minimum, paired with increased mammalian-cell expression compared to a reference mRNA having a higher uracil-content level (about 190% to about 200% of the theoretical minimum).

Stated Advantages

Documented Applications