Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

Abstract

Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

“…This finding is in contrast to the typical expectations for secondary structures in 5' UTRs to generally repress translation initiation. This finding is interesting because some synthetic small 5' UTR RNA hairpins have previously been found to improve protein expression 86 . In sum, our sequence selection strategy formalizes previously predicted rules for 5' UTR sequences that optimize ribosome load, and motivates an integrated approach to optimization of protein expression that jointly leverages our ribosome load dataset (Fig.…”

Factors Affecting the Expression of Recombinant Protein and Improvement Strategies in Chinese Hamster Ovary Cells

“…Gene sequence optimization can also adjust the GC content of genes, avoid base duplications, and eliminat restriction enzyme recognition sites, and also avoid similarity to important RNA motifs located in open reading frames that may interfere with mRNA processing and translational function, because they may cause ribosome pausing, in addition to factors such as CpG content and TATA boxes. Another 5′ untranslated region (5′ UTR) insertion into the RNA hairpin structure, the “regulatory element (RGE)”, also improves expression ( Eisenhut et al, 2020 ). In addition to gene sequence optimization, sequence motifs such as complementarity determining region three influences the rate of LC-HC dimerization during MAb synthesis, results in product-specific difference of yield ( Pybus et al, 2014 ).…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…The regulation of translation level and processing efficiency of translation products will also significantly affect the expression of target gene. Eisenhut et al developed the 5′-untranslated region (UTR) RNA-structures to impact translation efficiency, further systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression ( Eisenhut et al, 2020 ). Vivirius et al found a universal translation enhancer element in the 5′-end non-translation area of Hsp70 mRNA, which can enhance the translation efficiency of the cap-dependent structure ( Vivinus et al, 2001 ).…”