Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies

Abstract

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX

“…The complete ribosome profiling pipeline analysis is described in Figure 1: Sequencing data were pre-processed and aligned as described by Alexaki et al 5 as well as the step by step guide found in the README.txt accessible on GitHub.…”

“…In doing so, the amino acid sequence of the protein remains unaltered, therefore these changes were assumed to be inconsequential for the structure and function of the protein. However, this is not always true; through our ribosome profiling study, we described that these synonymous changes drastically altered translational kinetics and led to protein conformational changes 5 .…”

“…The ribosome profiling (footprinting) technique has only been around for a decade 1 but has already contributed tremendously to our understanding of translation efficiency and kinetics. Initially developed to systematically monitor protein translation in yeast 1 , it has since been adapted to work in a range of organisms 2,3 and to tackle a variety of questions. Ribosome profiling data typically consist of a set of sequences of ribosome protected fragments (RPF), designated as Ribo-seq data, which is accompanied by sequences from total RNA (RNA-seq).…”

“…We recently conducted a ribosome profiling study to examine the translation kinetics of blood coagulation factor IX 5 , a protein with great pharmaceutical interest. Two human embryonic kidney 293T (HEK293T) cell lines were lentivirally transduced, one with the wild type (WT) version of the gene and one with a codon optimized (CO) F9 5 . Codon optimization is a widely used technique that aims at increasing the protein expression levels by replacing multiple codons within a coding sequence with synonymous ones.…”

“…The translational kinetics of the F9 variants, along with the control genes, GAPDH and ACTB, were analyzed in detail in the original publication 5 . Similarly, any other gene of interest can be investigated in this dataset in terms of their rate of synthesis and translational kinetics; genes in the entire transcriptome can be compared to each other.…”