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\begin{document}

\title{Enhancing recombinant protein and viral vector production in mammalian
cells by targeting the YTHDF readers of N6--methyladenosine in mRNA}



\author[1]{Nga   Lao}%
\author[1]{Niall Barron}%
\affil[1]{National Institute for Bioprocessing Research and Training}%


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  \date{June 15, 2024}


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\begin{abstract}
N6--methyl adenosine (m6A) is the most abundant internal modification on
eukaryotic mRNA and has been implicated in a wide range of fundamental
cellular processes. This modification is regulated and interpreted by a
set of writer, eraser, and reader proteins. To date, there have been no
reports on the potential of mRNA epigenetic regulators to influence
recombinant protein expression in mammalian cells. In this study we
evaluated the potential of manipulating the expression of the m6A YTH
domain-containing readers, YTHDF1, 2, and 3 to improve recombinant
protein yield based on their role in regulating mRNA stability and
promoting translation. Using siRNA-mediated gene depletion, cDNA
over-expression and methylation-specific RNA immunoprecipitation, we
demonstrate that (i) knock-down of YTHDF2 enhances
(\textasciitilde{}2-fold) the levels of recombinant protein derived from
GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2
depletion on transgene expression is m6A-mediated and (iii) YTHDF2
depletion or over-expression of YTHDF1 increases viral protein
expression and yield of infectious lentiviral particles
(\textasciitilde{}2-3 fold) in HEK293 cells. We conclude that various
transgenes can be subjected to regulation by m6A regulators in mammalian
cell lines and that these findings demonstrate the utility of
epi-transcriptomic-based approaches to host cell line engineering for
improved recombinant protein and viral vector production.%
\end{abstract}%



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