Mammalian polycistronic expression systems for direct translation from RNA and cargo secretion.

Abstract

Disclosed herein are methods, compositions, and kits that enable the expression of multiple payload proteins from a single mRNA configured to achieve a predetermined stoichiometric ratio of a first-unit payload protein and a second-unit payload protein in a cell or cell-like environment. In some embodiments, nucleic acid compositions are provided that include a polynucleotide comprising a first nucleic acid unit (encoding one or more first-unit payload proteins) and a second nucleic acid unit (encoding one or more second-unit payload proteins). Each of the first and second nucleic acid units may include an engineered translation initiation site (eTIS) comprising a tunable element consisting of three nucleotides immediately upstream of the start codon. In some embodiments, the nucleic acid composition comprises at least one modified nucleotide and / or at least one nucleotide analog or nucleotide derivative. In some embodiments, the polynucleotide comprises a disclosed eTIS combination.

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