Structural, functional, and immunogenicity implications of F9 gene recoding.

Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX.

Ribosome profiling provides the opportunity to evaluate translation kinetics at codon level resolution. Here, we describe ribosome profiling data, generated from two HEK293T cell lines. The ribosome profiling data are composed of Ribo-seq (mRNA sequencing data from ribosome protected fragments) and RNA-seq data (total RNA sequencing). The two HEK293T cell lines each express a version of the F9 gene, both of which are translated into identical proteins in terms of their amino acid sequences. However, these F9 genes vary drastically in their codon usage and predicted mRNA structure. We also provide the pipeline that we used to analyze the data. Further analyzing this dataset holds great potential as it can be used i) to unveil insights into the composition and regulation of the transcriptome, ii) for comparison with other ribosome profiling datasets, iii) to measure the rate of protein synthesis across the proteome and identify differences in elongation rates, iv) to discover previously unidentified translation of peptides, v) to explore the effects of codon usage or codon context in translational kinetics and vi) to investigate cotranslational folding. Importantly, a unique feature of this dataset, compared to other available ribosome profiling data, is the presence of the F9 gene in two very distinct coding sequences.

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

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.

Translational and transcriptional responses in human primary hepatocytes under hypoxia.

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes. NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.

A Novel Strategy to Reverse General Anesthesia by Scavenging with the Acyclic Cucurbit[n]uril-type Molecular Container Calabadion 2.

BACKGROUND: Calabadion 2 is a new drug-encapsulating agent. In this study, the authors aim to assess its utility as an agent to reverse general anesthesia with etomidate and ketamine and facilitate recovery. METHODS: To evaluate the effect of calabadion 2 on anesthesia recovery, the authors studied the response of rats to calabadion 2 after continuous and bolus intravenous etomidate or ketamine and bolus intramuscular ketamine administration. The authors measured electroencephalographic predictors of depth of anesthesia (burst suppression ratio and total electroencephalographic power), functional mobility impairment, blood pressure, and toxicity. RESULTS: Calabadion 2 dose-dependently reverses the effects of ketamine and etomidate on electroencephalographic predictors of depth of anesthesia, as well as drug-induced hypotension, and shortens the time to recovery of righting reflex and functional mobility. Calabadion 2 displayed low cytotoxicity in MTS-3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-based cell viability and adenylate kinase release cell necrosis assays, did not inhibit the human ether-à-go-go-related channel, and was not mutagenic (Ames test). On the basis of maximum tolerable dose and acceleration of righting reflex recovery, the authors calculated the therapeutic index of calabadion 2 in recovery as 16:1 (95% CI, 10 to 26:1) for the reversal of ketamine and 3:1 (95% CI, 2 to 5:1) for the reversal of etomidate. CONCLUSIONS: Calabadion 2 reverses etomidate and ketamine anesthesia in rats by chemical encapsulation at nontoxic concentrations.