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Results for “codon optimization”

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PubMed2025

Assessment of different promoters in lentiviral vectors for expression of the N-acetyl-galactosamine-6-sulfate sulfatase gene.

Mucopolysaccharidosis IVA (MPS IVA) is caused by pathogenic variants in the GALNS gene encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycan (GAG) accumulation in multiple tissues, resulting in progressive skeletal dysplasia and poor quality of life. There is currently no effective treatment for this skeletal disease. This study proposes a novel lentiviral vector (LV)-based gene therapy that produces and secretes the active GALNS enzyme at supraphysiologic levels within the cells. LVs carrying the native GALNS encoding sequence (cDNA) were made under three different promoters: CBh, COL2A1, and CD11b. Moreover, we designed LVs carrying the native GALNS cDNA tagged with D8 octapeptide under the CD11b promoter and a human codon-optimized GALNS cDNA under the CBh promoter, respectively. Transduced HEK293 cells, HepG2 cells, and MPS IVA fibroblasts and chondrocytes were cultured for 8 and 30 days, and the media were collected every three days. The enzyme activity, GAG levels, and vector copy numbers (VCNs) in these cells and media were analyzed. LV with the COL2A1 promoter produced the highest enzyme activity in HEK293, HepG2, MPS IVA fibroblasts, and chondrocytes, followed by LV with the CBh promoter. VCNs were higher in MPS IVA fibroblasts treated with LV-CBh-hGALNS and in HepG2 cells treated with LV-CD11b-hGALNS than in HEK293 cells. Accumulated GAGs were normalized to wild-type levels by the LV gene therapy, especially with CBh and COL2A1 promoters. These findings, if further validated, could significantly impact the treatment of MPS IVA, offering a more effective and feasible treatment option.

Humans↗

PubMed2019

Cold chain and virus-free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes.

To prevent vaccine-associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype-2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus-free and cold chain-free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low-cost, cold chain/poliovirus-free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non-toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site-specific integration of CTB-VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9-15-fold in chloroplasts. GM1-ganglioside receptor-binding ELISA confirmed pentamer assembly of CTB-VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1-VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB-VP1co, formulated with plant-derived oral adjuvants, enhanced VP1-specific IgG1, VP1-IgA titres and neutralization (80%-100% seropositivity of Sabin-1, 2, 3). In contrast, IPV single dose resulted in <50% VP1-IgG1 and negligible VP1-IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB-VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low-cost solution to eradicate polio.

Animals↗

PubMed2023

Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by the absence of dystrophin, a membrane-stabilizing protein encoded by the DMD gene. Although mouse models of DMD provide insight into the potential of a corrective therapy, data from genetically homologous large animals, such as the dystrophin-deficient golden retriever muscular dystrophy (GRMD) model, may more readily translate to humans. To evaluate the clinical translatability of an adeno-associated virus serotype 9 vector (AAV9)-microdystrophin (μDys5) construct, we performed a blinded, placebo-controlled study in which 12 GRMD dogs were divided among four dose groups [control, 1 × 1013 vector genomes per kilogram (vg/kg), 1 × 1014 vg/kg, and 2 × 1014 vg/kg; n = 3 each], treated intravenously at 3 months of age with a canine codon-optimized microdystrophin construct, rAAV9-CK8e-c-μDys5, and followed for 90 days after dosing. All dogs received prednisone (1 milligram/kilogram) for a total of 5 weeks from day -7 through day 28. We observed dose-dependent increases in tissue vector genome copy numbers; μDys5 protein in multiple appendicular muscles, the diaphragm, and heart; limb and respiratory muscle functional improvement; and reduction of histopathologic lesions. As expected, given that a truncated dystrophin protein was generated, phenotypic test results and histopathologic lesions did not fully normalize. All administrations were well tolerated, and adverse events were not seen. These data suggest that systemically administered AAV-microdystrophin may be dosed safely and could provide therapeutic benefit for patients with DMD.

Animals↗

PubMed2025

BCKDHA-BCKDHB digenic gene therapy restores metabolic homeostasis in two mouse models and a calf with classic maple syrup urine disease.

Classic maple syrup urine disease (MSUD) results from biallelic mutations in genes that encode the branched-chain α-ketoacid dehydrogenase E1α (BCKDHA), E1β (BCKDHB), or dihydrolipoamide branched-chain transacylase (DBT) subunits, which interact to form the mitochondrial BCKDH complex that decarboxylates ketoacid derivatives of leucine, isoleucine, and valine. MSUD is an inborn error of metabolism characterized by recurrent life-threatening neurologic crises and progressive brain injury that can only be managed with an exacting prescription diet or allogeneic liver transplant. To develop a gene replacement therapy for MSUD, we designed a dual-function recombinant adeno-associated virus serotype 9 (rAAV9) vector to deliver codon-optimized BCKDHA and BCKDHB (rAAV9.hA-BiP-hB) to the liver, muscle, heart, and brain. rAAV9.hA-BiP-hB restored coexpression of BCKDHA and BCKDHB as well as BCKDH holoenzyme activity in BCKDHA-/- HEK293T cells and did not perturb physiologic branched-chain amino acid homeostasis in wild-type mice at a systemic dose of 2.7 × 1014 vector genomes per kilogram. In two models of severe MSUD (Bckdha-/- and Bckdhb-/- mice) and a newborn calf homozygous for BCKDHA c.248C>T, one postnatal injection prevented perinatal death, normalized growth, restored coordinated expression of BCKDHA and BCKDHB in the skeletal muscle, liver, heart, and brain, and stabilized MSUD biomarkers in the face of high protein ingestion. In summary, we developed a one-time BCKDHA-BCKDHB systemic dual-gene replacement strategy that holds promise as a therapeutic alternative to prescription diet and liver transplant for treatment of MSUD types 1A and 1B, the two most common forms of MSUD in humans.

Animals↗

PubMed1992

Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene of Escherichia coli.

Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. The structural gene (coaA) located at 90 min of the Escherichia coli chromosome was cloned and sequenced. The coaA gene was transcribed in the opposite direction to the flanking genes birA and thrU and produced a single 1.1-kb transcript. Translation of the coaA gene produced two protein products (36.4 and 35.4 kDa) that differed by eight amino acids at the amino terminus. The poor homology of the coaA promoter region to consensus E. coli promoter sequences and the low frequency of optimal codon usage (0.565) were consistent with the low abundance of pantothenate kinase. Strains containing multiple copies of the coaA gene possessed 76-fold-higher specific activity of pantothenate kinase; however, there was only a 2.7-fold increase in the steady-state level of CoA. These data corroborate the conclusion that regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration.

Amino Acid Sequence↗

PubMed2024

Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.

Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.

Cryptococcus neoformans↗

PubMed2025

Codon Composition in Human Oocytes Reveals Age-Associated Defects in mRNA Decay.

Oocytes from women of advanced reproductive age exhibit diminished developmental potential, but the underlying mechanisms remain incompletely defined. Oocyte maturation depends on translational control of maternal mRNA synthesized during growth. We performed a computational analysis on human oocytes from women <30 versus ≥40 years and observed that mRNA GC content correlates negatively with half-life in oocytes from young (<30 yr) but positively with oocytes from aged (>40 yr) women. In young oocytes, longer mRNA half-life is associated with lower protein abundance, whereas in aged oocytes GC content correlates positively with protein abundance. During the GV-to-MII transition, codon composition stratifies stability: codons that support rapid translation (optimal) stabilize mRNA, while slow-translating codons (non-optimal) promote decay. With reproductive aging, GC-containing codons become more optimal and align with increased protein abundance. These findings indicate that reproductive aging remodels codon-optimality-linked, translation-coupled mRNA decay, stabilizing a subset of GC-rich maternal mRNA that may be prone to excess translation during maturation. Our analysis is explicitly within human reproductive aging; it does not revisit cross-species stability rules. Instead, it shows that sequence-stability relations are reprogrammed with age within human oocytes, including an inversion of the GC-stability association during GV-to-MII transition. Disruption of the normal mRNA clearance program in aged oocytes may compromise oocyte competence and alter maternal mRNA dosage, with downstream consequences for early embryonic development.

Humans↗

PubMed2025

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

RNA Stability↗

PubMed1992

Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis.

In random mutagenesis, synthesis of an NNN triplet (i.e. equiprobable A, C, G, and T at each of the three positions in the codon) could be considered an optimal nucleotide mixture because all 20 amino acids are encoded. NN(G,C) might be considered a slightly more intelligent "dope" because the entire set of amino acids is still encoded using only half as many codons. Using a general algorithm described herein, it is possible to formulate more complex doping schemes which encode specific subsets of the twenty amino acids, excluding others from the mix. Maximizing the equiprobability of amino acid residues contributing to such a subset is suggested as an optimal basis for performing semi-random mutagenesis. This is important for reducing the nucleotide complexity of combinatorial cassettes so that "sequence space" can be searched more efficiently. Computer programs have been developed to provide tables of optimized dopes compatible with automated DNA synthesizers.

Amino Acid Sequence↗

PubMed1992

Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.

Base Sequence↗

PubMed2026

Multilayered nucleotide organization reveals purifying selection and host-driven adaptation in CPV and FPV.

Since feline panleukopenia virus (FPV) is considered the most likely ancestor of canine parvovirus (CPV), comprehensive comparisons of nucleotide organization in corresponding viral genes between CPV and FPV may provide novel insights into the evolutionary dynamics underlying the divergence of these two viruses. Here, we characterize the evolutionary patterns of CPV and FPV genes across multiple levels of nucleotide organization. Both viruses exhibited highly conserved nucleotide usage at nonsynonymous sites, with Ka/Ks patterns consistent with strong purifying selection, whereas synonymous sites showed greater variability. CpG dinucleotides were markedly underrepresented across all four viral genes, suggesting host-associated selective pressure and/or intrinsic nucleotide compositional constraints. Extensive nonrandom biases in synonymous codon usage, codon neighboring nucleotide context, and codon pair usage further revealed fine-scale genomic optimization shaped by natural selection and nucleotide compositional constraints. Structural protein genes (VP1 and VP2) displayed stronger codon usage bias and higher tRNA adaptation than nonstructural genes. Moreover, CPV genes showed greater translational adaptation to feline hosts than to canine hosts. These findings highlight how closely related parvoviruses exploit flexible nucleotide organization to facilitate host adaptation while maintaining essential protein functions.

Animals↗

PubMed2026

The genetic code at the balance point of error and demand.

The origin and organizing principles of the genetic code remain central problems in molecular evolution. The low probability of the natural codon-to-amino acid mapping arising by chance has spurred the hypothesis that its structure is optimized for robustness to mutations and translational errors. For the construction of effective molecular machines, the repertoire of encoded amino acids must also be diverse enough in physicochemical features. Here, we examine whether the standard genetic code can be understood as a near-optimal solution balancing these two objectives: minimizing error load and aligning codon assignments with the naturally occurring amino acid composition. Using simulated annealing, we explore this trade-off across a broad range of parameters. We find that the standard genetic code resides near an optimum in the fitness landscape of possible genetic codes. The degeneracy of the code plays a dual role, minimizing mistranslation errors while matching codon multiplicity to amino acid usage frequencies. As a result, uniform codon usage alone is sufficient to recover the empirical amino acid composition, without any additional bias. It is a highly effective solution that balances fidelity against resource availability constraints. A comparative analysis of natural variants also reveals a functional decoupling: error robustness acts as a rigid global constraint determined by code topology, whereas compositional alignment serves as a more flexible variable that adapts to lineage-specific demands. These results support a multi-objective optimization framework in which the genetic code reflects a balance between translational fidelity and proteomic demand.

Genetic Code↗

PubMed1978

Streptomycin causes misreading of natural messenger by interacting with ribosomes after initiation.

The induction of misreading by streptomycin in vitro, previously observed with synthetic messengers, is now demonstrated with natural (endogenous or viral) messenger by the use of extracts of temperature sensitive mutants lacking Glu--tRNA or Val--tRNA synthetase. With chain-elongating but noninitiating ribosomes (i.e., purified polysomes) deprived of an aminoacyl--tRNA, streptomycin and other aminoglycosides, over a wide range of concentrations, stimulate incorporation. With ribosomes initiating in the presence of streptomycin stimulation is also observed but it is restricted, just like phenotypic suppression in cells, to very low streptomycin concentrattions which evidently allow some ribosomes to initiate and later encounter them in the course of chain elongation. The stimulation is accompanied by an increase in the size of the products; hence, it is evidently due to substitution of an incorrect aminoacyl--tRNA for a missing one. The test introduced here also has revealed a misreading effect of streptomycin on resistant ribosomes. In addition, significant intrinsic misreading was observed without streptomycin, indicating that under optimal conditions for in vitro protein synthesis an empty codon is frequently read by an incorrect aminoacyl--tRNA.

Anti-Bacterial Agents↗

PubMed1975

Detecting evolutionary trends from molecular data. 1. Some measures of compositional nonrandomness.

The measures of compositional nonrandomness to be discussed as to their physical significance and to their power of detecting evolutionary significant variations are (see article)(pi a priori probability for amino acid i, ni its number of occurrences in a protein of length L). As a concrete example, the pi are here supposed to represent equal frequencies of all non-stop codons. For each quantity, four levels are defined: The base level, with optimal (i.e. minimal nonrandomness) composition, admitting non-integer values of ni; the integer level with optimal integer composition; the noise level, represented by a typical random cain; and the real protein level. On all these levels, S, which is the measure with the most direct physical sense, shows the smoothest behavior with the smallest relative fluctuations and thus the highest resolution.

Albumins↗

PubMed1992

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

Bacillus subtilis↗

PubMed2026

The Use of Deep Learning in RNA Therapeutic Development.

Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

Deep Learning↗

PubMed1992

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.

Amino Acid Sequence↗

PubMed1992

Construction and expression of nonsense suppressor tRNAs which function in plant cells.

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.

Anticodon↗