# Synthetic G-quadruplex components for predictable, precise two-level control of mammalian recombinant protein expression

## TL;DR

This paper introduces synthetic G-quadruplex components that allow precise and predictable control of protein expression in mammalian cells, useful for biopharmaceuticals and gene therapy.

## Contribution

The novel contribution is the development of synthetic G-quadruplex elements for two-level, tunable control of recombinant protein expression.

## Key findings

- Synthetic G-quadruplex elements were designed to regulate protein expression over two orders of magnitude.
- These components function predictably in different cell hosts and in both DNA and RNA contexts.
- A vector design platform was created to optimize multichain protein expression ratios efficiently.

## Abstract

Control of mammalian recombinant protein expression underpins the in vitro manufacture and in vivo performance of all biopharmaceutical products. However, routine optimization of protein expression levels in these applications is hampered by a paucity of genetic elements that function predictably across varying molecular formats and host cell contexts. Herein, we describe synthetic genetic components that are specifically built to simplify bioindustrial expression cassette design processes. Synthetic G-quadruplex elements with varying sequence feature compositions were systematically designed to exhibit a wide range of regulatory activities and inserted into identified optimal positions within a standardized, bioindustry compatible core promoter-5′UTR control unit. The resulting library tuned protein production rates over two orders of magnitude, where DNA and RNA G-quadruplexes could be deployed individually or in combination to achieve synergistic two-level regulatory control. We demonstrate these components can predictably and precisely tailor protein expression levels in (i) varying gene therapy and biomanufacturing cell hosts and (ii) both plasmid DNA and synthetic messenger RNA contexts. As an exemplar use case, a vector design platform was created to facilitate rapid optimization of polypeptide expression ratios for difficult-to-express multichain products. Permitting simple, predictable titration of recombinant protein expression, this technology should prove useful for gene therapy and biopharmaceutical manufacturing applications.

Graphical Abstract