Using highly expressed circular RNAs to substantially enhance protein expression yields in mammalian cells - SUMMARY
Large-scale production of recombinant proteins in mammalian cells is the process for manufacturing protein
biologics as medical therapies, for obtaining sufficient quantities of proteins for large-scale screens, and for
creating protein bioconjugates. Many proteins must be made in mammalian cells (as opposed to bacteria) if
the protein requires specific patterns of glycosylation, disulfide bond formation, or other modifications that can
only be formed in mammalian cells. However, protein production in mammalian cells is much less efficient
than protein production in bacterial cells. This is largely because the mRNA encoding the transgene is only a
small fraction of the total cellular mRNA in mammalian cells. In contrast, in bacterial cells, the transgene
mRNA can account for up to 50% of total cellular mRNA. In this application, we describe a strategy to increase
transcript levels in mammalian cells by 50-100-fold compared to current levels. This quantum leap in transcript
expression levels could fundamentally change protein production in mammalian cells. To do this, we will use
the novel “Tornado” technology for expressing transgenes as circular RNAs. These circular RNAs are
exceptionally stable and can accumulate to exceptionally high levels compared to corresponding linear
mRNAs. Chimerna scientists have generated key proof-of-concept data demonstrating the ability to express
RNA circles containing inserts similar in size to protein-coding open reading frames. In order to develop a
fundamentally new way to express proteins in mammalian cells at high levels, the specific aims of this proposal
are: (1) To optimize protein expression from Tornado-derived circular RNAs. Circular RNAs can encode
proteins if they contain an internal ribosome entry site (IRES). The goal of this aim is to characterize the
optimal IRES, insert size, and cell line suitable for protein translation using Tornado-expressed circular RNA.
We will systematically test each of these features and characterize protein output. (2) To compare protein
output from Tornado-derived circular RNA and linear mRNAs. In this aim, we will compare protein output
for cytosolic proteins and secreted proteins. We will directly compare linear to Tornado-encoded circular RNA
and determine if the high-level circular RNA production achieved using the Tornado system results in
increased protein production in cultured cells. Together, these experiments will allow us to test the idea that
genetically encoded circular RNAs can serve as a new platform for high-level protein expression in mammalian
cells. This expression system could have a major effect on biomedical research and protein manufacturing by
reducing costs for protein manufacturing, increasing protein yields and simplifying protein expression.
