tRNA Concentration Research Articles

Human selenocysteine synthase, SEPSECS, has evolved to optimize binding of a tRNA-based substrate.

The evolution of the genetic code to incorporate selenocysteine (Sec) enabled the development of a selenoproteome in all domains of life. O-phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal reaction of Sec synthesis on tRNASec in archaea and eukaryotes. Despite harboring four equivalent active sites, human SEPSECS binds no more than two tRNASec molecules. Though, the basis for this asymmetry remains poorly understood. In humans, an acidic, C-terminal, α-helical extension precludes additional tRNA-binding events in two of the enzyme monomers, stabilizing the SEPSECS•tRNASec complex. However, the existence of a helix exclusively in vertebrates raised questions about the evolution of the tRNA-binding mechanism in SEPSECS and the origin of its C-terminal extension. Herein, using a comparative structural and phylogenetic analysis, we show that the tRNA-binding motifs in SEPSECS are poorly conserved across species. Consequently, in contrast to mammalian SEPSECS, the archaeal ortholog cannot bind unacylated tRNASec and requires an aminoacyl group. Moreover, the C-terminal α-helix 16 is a mammalian innovation, and its absence causes aggregation of the SEPSECS•tRNASec complex at low tRNA concentrations. Altogether, we propose SEPSECS evolved a tRNASec binding mechanism as a crucial functional and structural feature, allowing for additional levels of regulation of Sec and selenoprotein synthesis.

A highly sensitive fluorescence sensor for tobacco mosaic virus RNA based on DSN cycle and ARGET ATRP double signal amplification

AbstractEarly and sensitive detection of tobacco mosaic virus (TMV) is of great significance for improving crop yield and protecting germplasm resources. Herein, we constructed a novel fluorescence sensor to detect TMV RNA (tRNA) through double strand specific nuclease (DSN) cycle and activator regenerative electron transfer atom transfer radical polymerization (ARGET ATRP) dual signal amplification strategy. The hairpin DNA complementarily paired with tRNA was used as a recognition unit to specifically capture tRNA. By the double‐stranded DNA hydrolyzed with DSN, tRNA is released to open more hairpin DNA, and more complementary DNA (cDNA) is bound to the surface of the magnetic beads (MBs) to achieve the first amplification. After binding with the initiator, the cDNA employed ARGET ATRP to attach more fluorescent signal molecules to the surface of MBs, thus achieving the second signal amplification. Under the optimal experimental conditions, the logarithm of fluorescence intensity versus tRNA concentration showed a good linear relationship in the range of 0.01–100 pM, with a detection limit of 1.03 fM. The limit of detection (LOD) was calculated according to LOD = 3 N/S. Besides, the sensor showed good reproducibility and stability, which present provided new method for early and highly sensitive detection for plant viruses.

Escherichia coli tRNA (Gm18) methyltransferase (TrmH) requires the correct localization of its methylation site (G18) in the D-loop for efficient methylation.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

Linear and nonlinear optical properties of transfer ribonucleic acid (tRNA) thin solid films.

We successfully obtained transfer ribonucleic acid (tRNA) thin solid films (TSFs) using an aqueous solution precursor in an optimized deposition process. By varying the concentration of RNA and deposition process parameters, uniform solid layers of solid RNA with a thickness of 30 to 46 nm were fabricated consistently. Linear absorptions of RNA TSFs on quartz substrates were experimentally investigated in a wide spectral range covering UV–VIS–NIR to find high transparency for λ > 350 nm. We analyzed the linear refractive indices, n(λ) of tRNA TSFs on silicon substrates by using an ellipsometer in the 400 to 900 nm spectral range to find a linear correlation with the tRNA concentration in the aqueous solution. The thermo-optic coefficient (dn/dT) of the films was also measured to be in a range −4.21 × 10−4 to −5.81 × 10−4 °C−1 at 40 to 90 °C. We furthermore characterized nonlinear refractive index and nonlinear absorption of tRNA TSFs on quartz using a Z-scan method with a femtosecond laser at λ = 795 nm, which showed high potential as an efficient nonlinear optical material in the IR spectral range.

Compelling Evidence Suggesting the Codon Usage of SARS-CoV-2 Adapts to Human After the Split From RaTG13.

SARS-CoV-2 needs to efficiently make use of the resources from hosts in order to survive and propagate. Among the multiple layers of regulatory network, mRNA translation is the rate-limiting step in gene expression. Synonymous codon usage usually conforms with tRNA concentration to allow fast decoding during translation. It is acknowledged that SARS-CoV-2 has adapted to the codon usage of human lungs so that the virus could rapidly proliferate in the lung environment. While this notion seems to nicely explain the adaptation of SARS-CoV-2 to lungs, it is unable to tell why other viruses do not have this advantage. In this study, we retrieve the GTEx RNA-seq data for 30 tissues (belonging to over 17 000 individuals). We calculate the RSCU (relative synonymous codon usage) weighted by gene expression in each human sample, and investigate the correlation of RSCU between the human tissues and SARS-CoV-2 or RaTG13 (the closest coronavirus to SARS-CoV-2). Lung has the highest correlation of RSCU to SARS-CoV-2 among all tissues, suggesting that the lung environment is generally suitable for SARS-CoV-2. Interestingly, for most tissues, SARS-CoV-2 has higher correlations with the human samples compared with the RaTG13-human correlation. This difference is most significant for lungs. In conclusion, the codon usage of SARS-CoV-2 has adapted to human lungs to allow fast decoding and translation. This adaptation probably took place after SARS-CoV-2 split from RaTG13 because RaTG13 is less perfectly correlated with human. This finding depicts the trajectory of adaptive evolution from ancestral sequence to SARS-CoV-2, and also well explains why SARS-CoV-2 rather than other viruses could perfectly adapt to human lung environment.

Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates

Variation in translation-elongation kinetics along a transcript’s coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, the presence of proline in the ribosome’s P- or A-site slows down translation, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized. Here, we study Saccharomyces cerevisiae using a combination of bioinformatics, mutational experiments, and evolutionary analyses, and show that many different pairs of amino acids and their associated tRNA molecules predictably and causally encode translation rate information when these pairs are present in the A- and P-sites of the ribosome independent of other factors known to influence translation speed including mRNA structure, wobble base pairing, tripeptide motifs, positively charged upstream nascent chain residues, and cognate tRNA concentration. The fast-translating pairs of amino acids that we identify are enriched four-fold relative to the slow-translating pairs across Saccharomyces cerevisiae’s proteome, while the slow-translating pairs are enriched downstream of domain boundaries. Thus, the chemical identity of amino acid pairs contributes to variability in translation rates, elongation kinetics are causally encoded in the primary structure of proteins, and signatures of evolutionary selection indicate their potential role in co-translational processes.

RNA-targeted Cu(II)-based potential antitumor drug entity: comprehensive structural, biological {DNA/RNA binding, cleavage, cytotoxicity} and computational studies

Copper-based bis(hydroxy naphthaldehyde) complex with axial mono aqua-coordination was synthesized and thoroughly characterized by various spectroscopic{IR, UV-vis, EPR}, ESI-Mass and single X-ray crystallographic studies. The single X-ray crystallography of the complex revealed the square pyramidal coordination geometry with P21 space group with axial water molecule ligated to copper centre. The geometry of the complex was further validated by DFT calculations, which was in accordance with other spectroscopic studies. The binding profile of the complex with ct-DNA and tRNA was carried out by employing various biophysical (absorption, fluorescence, circular dichroism, morphological studies) and computational studies (DFT, molecular docking). The experimental results revealed efficient binding of the complex with both ct-DNA and tRNA primarily, via non-covalent interactions. The binding studies Kb and K values revealed 10-fold greater binding affinity of the complex for tRNA as compared to ct-DNA. The in silico molecular docking further validated the interaction of complex within the hydrophobic pocket of ct-DNA and tRNA. The concentration and time dependent cleavage studies of DNA and tRNA were performed by employing gel electrophoresis assay. The cytotoxic activity of the complex was performed on a panel of human cell lines viz., leukemia (K-562), pancreatic (MIA-PA-CA-2), hepatoma (Hep-G2), cervical (HeLa), and breast (MDA-MB-231) by SRB assay. The complex exhibited selectively remarkably good cytotoxic potential on leukemia (K-562), cervical (HeLa) and hepatoma (Hep-G2) cancer cell lines. Communicated by Ramaswamy H. Sarma

Codon and amino acid content are associated with mRNA stability in mammalian cells.

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in the cell and is a major control point for modulating gene expression. In yeast and other model organisms, codon identity is a powerful determinant of transcript stability, contributing broadly to impact half-lives. General principles governing mRNA stability are poorly understood in mammalian systems. Importantly, however, the degradation machinery is highly conserved, thus it seems logical that mammalian transcript half-lives would also be strongly influenced by coding determinants. Herein we characterize the contribution of coding sequence towards mRNA decay in human and Chinese Hamster Ovary cells. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Surprisingly, however, we also observe that the amino acid content of a gene is an additional determinant correlating with transcript stability. The impact of codon and amino acid identity on mRNA decay appears to be associated with underlying tRNA and intracellular amino acid concentrations. Accordingly, genes of similar physiological function appear to coordinate their mRNA stabilities in part through codon and amino acid content. Together, these results raise the possibility that intracellular tRNA and amino acid levels interplay to mediate coupling between translational elongation and mRNA degradation rate in mammals.

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts.

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of invitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an invitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Efficiency of protein synthesis inhibition depends on tRNA and codon compositions

Regulation and maintenance of protein synthesis are vital to all organisms and are thus key targets of attack and defense at the cellular level. Here, we mathematically analyze protein synthesis for its sensitivity to the inhibition of elongation factor EF-Tu and/or ribosomes in dependence of the system’s tRNA and codon compositions. We find that protein synthesis reacts ultrasensitively to a decrease in the elongation factor’s concentration for systems with an imbalance between codon usages and tRNA concentrations. For well-balanced tRNA/codon compositions, protein synthesis is impeded more effectively by the inhibition of ribosomes instead of EF-Tu. Our predictions are supported by re-evaluated experimental data as well as by independent computer simulations. Not only does the described ultrasensitivity render EF-Tu a distinguished target of protein synthesis inhibiting antibiotics. It may also enable persister cell formation mediated by toxin-antitoxin systems. The strong impact of the tRNA/codon composition provides a basis for tissue-specificities of disorders caused by mutations of human mitochondrial EF-Tu as well as for the potential use of EF-Tu targeting drugs for tissue-specific treatments.

Towards a New Aminoacylation Assay for Alanyl‐tRNA Synthetases

Aminoacyl‐tRNA synthetases are crucial enzymes in protein biosynthesis, ensuring the fidelity of the genetic code by charging transfer RNAs with the appropriate amino acid. Aminoacylation is typically measured using radioactively labeled substrates, using assays that are non‐continuous, relatively expensive, and time consuming. Recently, a new assay1 was developed for tyrosyl‐tRNA synthetases in which the formation of AMP during the aminoacylation reaction is coupled to production of NADH which can be measured spectrophotometrically. To improve sensitivity, the aminoacyl‐tRNA product was regenerated through hydrolysis with a second enzymatic activity, allowing detection of aminoacylation even at low concentrations of tRNA. We have extended this assay to the alanyl‐tRNA synthetase system. Three different aminoacylation and tRNA regeneration schemes have been developed – mischarging with serine, followed by hydrolysis with the intrinsic editing domain; mischarging with glycine, followed by hydrolysis with D‐aminoacyl‐tRNA deacylase (DTD); and charging with alanine, followed by hydrolysis with a variant of DTD.Support or Funding InformationCarleton College and the Towsley FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of tRNA-mediated +1 ribosomal frameshifting

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA-mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

Codon usage revisited: Lack of correlation between codon usage and the number of tRNA genes in enterobacteria

It is widely believed that if a high number of genes are found for any tRNA in a rapidly replicating bacteria, then the cytoplasmic levels of that tRNA will be high and an open reading frame containing a higher frequency of the complementary codon will be translated faster. This idea is based on correlations between the number of tRNA genes, tRNA concentration and the frequency of codon usage observed in a limited number of strains as well as from the fact that artificially changing the number of tRNA genes alters translation efficiency and consequently the amount of properly folded protein synthesized. tRNA gene number may greatly vary in a genome due to duplications, deletions and lateral transfer which in turn would alter the levels and functionality of many proteins. Such changes are potentially deleterious for fitness and as a result it is expected that changes in tRNA gene numbers should be accompanied by a modification of the frequency of codon usage. In contrast to this model, when comparing the number of tRNA genes and the frequency of codon usage of several Salmonella enterica and Escherichia coli strains we found that changes in the number of tRNA genes are not correlated to changes in codon usage. Furthermore, these changes are not correlated with a change in the efficiency of codon translation. These results suggest that once a genome gains or loses tRNA genes, it responds by modulating the concentrations of tRNAs rather than modifying its frequency of codon usage.

Evolutionarily-Encoded Translation Kinetics Coordinate Co-Translational SSB Chaperone Binding in Yeast

Chaperones can bind to the ribosomes and nascent polypeptides co-translationally to assist protein folding. It was not known previously whether there is any interplay between chaperone binding and translation kinetics. To study this effect, we utilize the high-throughput transcriptome-wide quantitative data for translation kinetics and chaperone binding from ribosome profiling and selective ribosome profiling methods respectively. In vivo selective ribosome profiling has shown that yeast Hsp70 chaperone Ssb associates broadly with a major fraction of nascent proteins. Using the ribosome footprint densities as a measure of local translation rate, we find that mRNA segments within the ribosome are translated faster during periods of Ssb binding to the nascent polypeptides compared to when Ssb is not bound. The acceleration of translation is maintained even when Ssb is knocked out thus implying an inherent encoding of faster translation within the mRNA sequence. Testing for mRNA features that can slow translation, we find that mRNA segments translated by Ssb-engaged ribosomes are enriched for fast-translated codons (having higher cognate tRNA concentrations), are depleted for slowly translated codons, and contain fewer proline codons. In addition, mRNA segments located 1-15 nucleotides downstream of Ssb-bound ribosomes have reduced mRNA secondary structure. Finally, nascent chain segments located in the ribosome tunnel of Ssb-bound ribosomes have average numbers of positively charged residues but are enriched in negatively charged residues. Taken together, these evolutionarily-encoded mRNA and nascent chain features cause faster translation during Ssb binding. This finding is significant as any alteration of translation kinetics due to synonymous mutations can potentially disrupt efficient binding of Ssb leading to possible misfolding of the protein without any change in its sequence.

Codon optimality, bias and usage in translation and mRNA decay.

The advent of ribosome profiling and other tools to probe mRNA translation has revealed that codon bias - the uneven use of synonymous codons in the transcriptome - serves as a secondary genetic code: a code that guides the efficiency of protein production, the fidelity of translation and the metabolism of mRNAs. Recent advancements in our understanding of mRNA decay have revealed a tight coupling between ribosome dynamics and the stability of mRNA transcripts; this coupling integrates codon bias into the concept of codon optimality, or the effects that specific codons and tRNA concentrations have on the efficiency and fidelity of the translation machinery. In this Review, we first discuss the evidence for codon-dependent effects on translation, beginning with the basic mechanisms through which translation perturbation can affect translation efficiency, protein folding and transcript stability. We then discuss how codon effects are leveraged by the cell to tailor the proteome to maintain homeostasis, execute specific gene expression programmes of growth or differentiation and optimize the efficiency of protein production.

How the Ribosomal A-Site Finger Can Lead to tRNA Species-Dependent Dynamics.

Proteins are synthesized by the joint action of the ribosome and tRNA molecules, where the rate of synthesis can be affected by numerous factors, such as the concentration of tRNA, the binding affinity of tRNA for the ribosome, or post-transcriptional modifications. Here, we expand this range of contributors by demonstrating how differences in tRNA structure can give rise to tRNA species-specific dynamics in the ribosome. To show this, we perform simulations of A/P hybrid-state formation for two tRNA species (tRNAPhe and tRNALeu), which differ in the size of their variable loops (VLs). These calculations reveal that steric interactions between the VL and the ribosomal A-site finger (ASF, i.e., H38 of 23S rRNA) can directly modulate the free-energy landscape for each tRNA species. We also find that tRNA and ASF motions are highly correlated, where fluctuations of the ASF are predictive of tRNA transition events. Finally, by introducing perturbations to the model, we demonstrate that ASF flexibility is a determinant of the rate of A/P hybrid-state formation.

A quantitative model for the rate-limiting process of UGA alternative assignments to stop and selenocysteine codons.

Ambiguity in genetic codes exists in cases where certain stop codons are alternatively used to encode non-canonical amino acids. In selenoprotein transcripts, the UGA codon may either represent a translation termination signal or a selenocysteine (Sec) codon. Translating UGA to Sec requires selenium and specialized Sec incorporation machinery such as the interaction between the SECIS element and SBP2 protein, but how these factors quantitatively affect alternative assignments of UGA has not been fully investigated. We developed a model simulating the UGA decoding process. Our model is based on the following assumptions: (1) charged Sec-specific tRNAs (Sec-tRNASec) and release factors compete for a UGA site, (2) Sec-tRNASec abundance is limited by the concentrations of selenium and Sec-specific tRNA (tRNASec) precursors, and (3) all synthesis reactions follow first-order kinetics. We demonstrated that this model captured two prominent characteristics observed from experimental data. First, UGA to Sec decoding increases with elevated selenium availability, but saturates under high selenium supply. Second, the efficiency of Sec incorporation is reduced with increasing selenoprotein synthesis. We measured the expressions of four selenoprotein constructs and estimated their model parameters. Their inferred Sec incorporation efficiencies did not correlate well with their SECIS-SBP2 binding affinities, suggesting the existence of additional factors determining the hierarchy of selenoprotein synthesis under selenium deficiency. This model provides a framework to systematically study the interplay of factors affecting the dual definitions of a genetic codon.

Protein Synthesis Times Scale with Gene Length because the Determinants of Translation Speed are Randomly Distributed Across Genes

Many of the molecular factors influencing codon translation speed have been identified, and their relative contributions estimated. These factors include tRNA concentration, the presence of charged nascent-chain residues in the ribosome exit tunnel, mRNA secondary structure, proline residues at the A or P sites of the ribosome and steric interactions between ribosomes translating a transcript. Here, we combine this information with genomic information from E. coli, yeast and humans in a simulation model of translation to estimate the synthesis time of cytopolasmic proteins. We find that regardless of the organism, the synthesis time of a protein scales linearly with the length of mRNA's coding sequence even though there is a large variation in the translation speed of individual codons. We demonstrate that this scaling arises because the molecular determinants of translation speed are distributed randomly across the mRNA transcripts and that this distribution is generated from a Poisson point process. As a consequence, the Law of Large Numbers is followed and for any given transcript fast-translating segments are canceled out by a similar number of slow-translating segments resulting a constant average codon translation rate between different transcripts. This means that a protein's average synthesis time can be accurately predicted based solely on the corresponding gene length, provided the average codon translation speed is known. Thus, although evolution has biased codon usage between different genes in an organism, there is still a large degree of randomness associated with how individual codon translation speeds have been distributed. These results also provide an explanation for the observation from ribosome profiling that different transcripts have the same average codon translation speed in mouse-stem cells.

Preferential use of minor codons in the translation initiation region of human genes.

More than 31,000 protein-coding sequences (CCDS) have been identified in the human genome. Here, we analyzed codon usage in all human CCDS and found that there is a preferential usage of minor codons for Ala (CGC), Pro (CCG), Ser (UCG), and Thr (ACG) in the initial 50-codon sequences of the CCDS. These codons, with consensus XCG sequences, are most infrequently used among their synonymous codons. Thus, the tRNA concentrations per codon are considered to be highest for the minor codons for Ala, Pro, Ser and Thr in comparison with other synonymous codons for each of them to enhance the translation efficiency. This suggests that human genes are regulated at the level of translation by preferentially using minor codons within the first 50 codons of the CCDS. This hypothesis was experimentally confirmed by comparing the expression of the luciferase gene encoded by minor codons with that encoded by major codons.

728. Tissue-Directed Transgene Engineering for AAV and Lentivector Gene Therapy Approaches

Clinical gene therapy frequently is encumbered by low transgene product biosynthesis at predictably safe vector doses. It has been hypothesized that the presence of rare codons may regulate transgene product expression through depletion of the available cognate tRNA pool. Codon optimization is the prominent strategy utilized to overcome this hypothesized limitation and involves replacing rare, presumably translation rate-limiting, codons with the most frequent ones. Typical algorithms attempt to match the codon usage frequency of the transgene coding sequence to that of target organism's total mRNA pool, which has been shown to approximate the overall available tRNA concentrations. Upon closer examination, it appears that both codon frequency and tRNA content vary between tissue types. Therefore, we hypothesize that codon-optimization can be improved by tailoring to the codon-frequency of the most highly expressed genes present in target cell types. Our tissue-directed codon optimization algorithm utilizes novel codon usage indices generated from target cell gene expression data. As proof of concept, we developed tissue-codon optimized variants of coagulation factor VIII (FVIII) to be utilized in lenti- and adeno-associated viral (LV and AAV, respectively) vectors. These two vectors are the leading platforms for clinical gene therapy of hemophilia A. LV is utilized to target autologous hematopoietic cell types ex vivo while AAV is delivered intravenously to genetically-modify hepatocytes. However to date, development-stage LV and AAV gene therapy products for hemophilia A have been characterized by low-level FVIII transgene product biosynthesis. Initially, we designed human hepatocyte-, monocyte- and standard overall human-optimized FVIII constructs (LCO, MCO and HCO, respectively) to be compared to wild-type FVIII. Upon initial examination, expression of LCO-FVIII was shown to be 3-fold greater than either MCO- or wild-type FVIII from the human hepatoma cell line, HepG2, following transient transfection. In contrast, LCO-FVIII and MCO-FVIII expression was diminished 12 and 4-fold, respectively, compared to wild-type in the ‘neutral’ human embryonic kidney 293T cell line. Furthermore, following hydrodynamic injection of naked plasmid DNA into hemophilia A mice, LCO-FVIII exhibited a sustained 10-fold increase in FVIII expression relative to the HCO-FVIII comparator. In attempt to generate a lead candidate for clinical translation, we utilized several of our most promising vector components to construct a liver-optimized AAV8 vector consisting of LCO-ET3, our previously described high-expression bioengineered FVIII variant, transcriptionally driven by a novel 146bp liver-directed promoter and adjacent MVM intron. Following intravenous delivery into hemophilia A mice, vector doses of 1e12 and 1e11 vector genomes per kg achieved sustained, predictable curative plasma FVIII levels of 200% and 20% of normal human levels, respectively. These initial results support the utility of our novel approach of clinical tissue-directed transgene optimization.