Genome Recoding Research Articles

Genomically recoded Escherichia coli with optimized functional phenotypes.

Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.

Breaking the Degeneracy of Sense Codons – How Far Can We Go?

AbstractGenetic code expansion aims to incorporate non‐canonical amino acids (ncAAs) into biological systems, enhancing protein functionality or enabling the in vitro selection of peptides from diverse mRNA displayed libraries. Typically, genetic code expansion has involved reassignment of stop codons to ncAAs through orthogonal translation systems. This review instead focuses on efforts to expand the genetic code by breaking the redundancy of sense codons in vitro and in vivo. In vivo, orthogonal aminoacyl‐tRNA synthetase (AARS)/tRNA/AA systems are able to compete with endogenous machinery, enabling partial to full codon reassignment. Recent approaches, like genome recoding, offer potential solutions to reduce competition. In vitro studies utilize cell extract‐based or reconstituted translation systems, allowing precise control of codon usage via gene design and tRNA addition, making breaking of sense degeneracy easier. In these systems several unsplit codon boxes have been successfully reassigned multiple to ncAAs. These efforts showcase both the successes and challenges in achieving orthogonality and selective codon decoding and point towards a future where the 64 codons can encode more than 30 monomers, enabling new advances in synthetic biology and drug discovery.

Syn-CpG-Spacer: A Panel web app for synonymous recoding of viral genomes with CpG dinucleotides.

Vertebrate genomes contain lower than expected frequencies of the CpG dinucleotide. Consequently, many vertebrate viruses have evolved to mimic this composition, possibly in order to evade host antiviral defences (Greenbaum et al., 2008). For example, the antiviral protein ZAP binds CpGs in viral single stranded RNA with specific spacing requirements (Gonçalves-Carneiro et al., 2022), though CpGs are also likely depleted in viral genomes due to other selective pressures (Forni et al., 2023). Increasing CpG abundance by synonymous recoding could facilitate attenuation of viruses without compromising their epitope antigenicity by changing non-CpG codons to alternatives containing CpG without changing the overall amino acid sequence (Gonçalves-Carneiro et al., 2022; Le Nouën et al., 2019; Sharp et al., 2023). There are three ways CpGs can be synonymously introduced in codons: at positions 1-2 for arginine (e.g. AGA → CGA), 2-3 for several amino acids (e.g. ACA → ACG), or in a 3-1 split configuration, if a subsequent codon begins with a G (e.g. ATA-GCA → ATC-GCA). Syn-CpG-Spacer is a Python progressive web app (PWA) (MDN Web Docs, 2023) made with the Panel library (Panel Development Team, 2024) that allows for consistent recoding of viral sequences and applying biologically relevant constraints. These include setting a minimum gap between CpG's, optimising for an average CpG gap, protecting cis-acting regulatory signals from modification, and modulating the A-content in the overall sequence. The app features a sequence viewer made with the Bokeh library (Bokeh Development Team, 2024) that highlights CpG dinucleotides, allowing for efficient analysis of the resulting distribution of CpGs. This is complemented by a statistical data table. Utilising Biopython (Cock et al., 2009) modules, the user can load their sequence as a FASTA file and download the outputs as an alignment in the same format. As a PWA running on Pyodide (The Pyodide development team, 2023), the code is only executed in the user's browser and they can install the app onto their machine for offline use.

Blastocrithidia nonstop mitochondrial genome and its expression are remarkably insulated from nuclear codon reassignment.

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

Flavivirus genome recoding by codon optimisation confers genetically stable in vivo attenuation in both mice and mosquitoes.

Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.

Novel approaches for the rapid development of rationally designed arbovirus vaccines

Vector-borne diseases, including those transmitted by mosquitoes, account for more than 17% of infectious diseases worldwide. This number is expected to rise with an increased spread of vector mosquitoes and viruses due to climate change and man-made alterations to ecosystems. Among the most common, medically relevant mosquito-borne infections are those caused by arthropod-borne viruses (arboviruses), especially members of the genera Flavivirus and Alphavirus. Arbovirus infections can cause severe disease in humans, livestock and wildlife. Severe consequences from infections include congenital malformations as well as arthritogenic, haemorrhagic or neuroinvasive disease. Inactivated or live-attenuated vaccines (LAVs) are available for a small number of arboviruses; however there are no licensed vaccines for the majority of these infections. Here we discuss recent developments in pan-arbovirus LAV approaches, from site-directed attenuation strategies targeting conserved determinants of virulence to universal strategies that utilize genome-wide re-coding of viral genomes. In addition to these approaches, we discuss novel strategies targeting mosquito saliva proteins that play an important role in virus transmission and pathogenesis in vertebrate hosts.For rapid pre-clinical evaluations of novel arbovirus vaccine candidates, representative in vitro and in vivo experimental systems are required to assess the desired specific immune responses. Here we discuss promising models to study attenuation of neuroinvasion, neurovirulence and virus transmission, as well as antibody induction and potential for cross-reactivity. Investigating broadly applicable vaccination strategies to target the direct interface of the vertebrate host, the mosquito vector and the viral pathogen is a prime example of a One Health strategy to tackle human and animal diseases.

CpG dinucleotide enrichment in the influenza A virus genome as a live attenuated vaccine development strategy.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. Problematically, recoding typically hinders virus growth, but this may be rectified using CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), and so in principle, removing ZAP sensing from a virus propagation system will reverse attenuation of a CpG-enriched virus, enabling high titre yield of a vaccine virus. We tested this using a vaccine strain of influenza A virus (IAV) engineered for increased CpG content in genome segment 1. Virus attenuation was mediated by the short isoform of ZAP, correlated with the number of CpGs added, and was enacted via turnover of viral transcripts. The CpG-enriched virus was strongly attenuated in mice, yet conveyed protection from a potentially lethal challenge dose of wildtype virus. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Unexpectedly, in both MDCK cells and embryonated hens' eggs that are used to propagate live attenuated influenza vaccines, the ZAP-sensitive virus was fully replication competent. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform to augment existing live attenuated vaccines.

Rational attenuation of RNA viruses with zinc finger antiviral protein

Attenuation of a virulent virus is a proven approach for generating vaccines but can be unpredictable. For example, synonymous recoding of viral genomes can attenuate replication but sometimes results in pleiotropic effects that confound rational vaccine design. To enable specific, conditional attenuation of viruses, we examined target RNA features that enable zinc finger antiviral protein (ZAP) function. ZAP recognized CpG dinucleotides and targeted CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP were undefined. Using synonymously mutated HIV-1 genomes, we defined several sequence features that govern ZAP sensitivity and enable stable attenuation. We applied rules derived from experiments with HIV-1 to engineer a mutant enterovirus A71 genome whose attenuation was stable and strictly ZAP-dependent, both in cell culture and in mice. The conditionally attenuated enterovirus A71 mutant elicited neutralizing antibodies that were protective against wild-type enterovirus A71 infection and disease in mice. ZAP sensitivity can thus be readily applied for the rational design of conditionally attenuated viral vaccines.

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida.

Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

Development of safe and highly protective live-attenuated SARS-CoV-2 vaccine candidates by genome recoding.

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We construct a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and assess their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses do not cause clinical symptoms but are highly immunogenic and induce strong protective immunity. Attenuated viruses replicate efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters develop no signs of disease and rapidly clear challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic.

Impact of Synonymous Genome Recoding on the HIV Life Cycle.

Synonymous mutations within protein coding regions introduce changes in DNA or messenger (m) RNA, without mutating the encoded proteins. Synonymous recoding of virus genomes has facilitated the identification of previously unknown virus biological features. Moreover, large-scale synonymous recoding of the genome of human immunodeficiency virus type 1 (HIV-1) has elucidated new antiviral mechanisms within the innate immune response, and has improved our knowledge of new functional virus genome structures, the relevance of codon usage for the temporal regulation of viral gene expression, and HIV-1 mutational robustness and adaptability. Continuous improvements in our understanding of the impacts of synonymous substitutions on virus phenotype – coupled with the decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments – have enhanced our ability to identify potential HIV-1 and host factors and other aspects involved in the infection process. In this review, we address how silent mutagenesis impacts HIV-1 phenotype and replication capacity. We also discuss the general potential of synonymous recoding of the HIV-1 genome to elucidate unknown aspects of the virus life cycle, and to identify new therapeutic targets.

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Development of Safe and Highly Protective Live-Attenuated SARS-CoV-2 Vaccine Candidates by Genome Recoding

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We constructed a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome, and assessed their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses did not cause clinical symptoms, but were highly immunogenic and induced strong protective immunity. Attenuated viruses replicated efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters developed no signs of disease and rapidly cleared challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic.Funding: This research was supported by the Deutsche Forschungsgemeinschaft (DFG), grant OS143/16-1 and COVID-19 grants from Freie Universität Berlin and Berlin University Alliance awarded to NO, the DFG grant SFB-TR84/Z01b awarded to ADG and JT and the SwissNational Science Foundation, grants 31CA30_196644, 31CA30_196062, and 310030_173085 awarded to VT.Conflict of Interest: The authors declare no competing interests.Ethical Approval: In vitro and animal work was done under biosafety conditions in the BSL-3 facility at the Institut für Virologie, Freie Universität Berlin, Germany. All animal experiments wereapproved by the Landesamt für Gesundheit und Soziales in Berlin, Germany (permit number0086/20) and done in compliance with relevant national and international guidelines for care and humane use of animals.

Genome recoding strategies to improve cellular properties: mechanisms and advances.

The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase–tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.

Genome recoding: a review of basic concepts, current research and future prospects of virus attenuation for controlling plant viral diseases

Plants are very susceptible to pathogens and every year, 25% of crop loss is caused by various types of pathogens including viruses. Many different strategies are being used for developing resistance against virus infection, including RNA silencing, and the genome editing including CRISPR-Cas-9 but these may produce variants/recombinants and could cause the problems for future crops. Another promising approach named as genome recoding or rewriting would be a better potential tool for controlling viral infections in plants. It relies on the concepts of replacement of synonymous codons, change in codon bias, codon pair bias and dinucleotide content. Recoding of the genome does not alter the amino acid sequences but it affects the expression level and translation efficiency. In the present report, the concept of synonymous codons, the basics of genome recoding and the possible strategies to generate genome recoded organisms are provided in details. Viral attenuation has been achieved by consideration of dinucleotide bias and codon pair bias manipulations and used in the synthesis of vaccines against various types of pathogenic bacteria and viruses. The idea of the future scope of genome recoding for developing virus-resistant plants and their challenges for the same are also comprehensively discussed. Although genome recoding is not yet tested on plants, however it could be very helpful in controlling plant viral diseases. So, it is a novel emerging area of research for developing viral resistant plants and thus would help in minimizing the agricultural losses in the near future.

Mechanism of Virus Attenuation by Codon Pair Deoptimization

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias canbe used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.

Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies.

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies.

Extensive genomic recoding by codon-pair deoptimization selective for mammals is a flexible tool to generate attenuated vaccine candidates for dengue virus 2

The four serotypes of dengue virus (DENV) are the leading etiologic agent of disease caused by arthropod-borne viruses (arboviruses) in the world, with billions at risk of DENV infection spread by infected mosquitoes. DENV causes illness ranging from dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV proliferates well in two different host systems, an invertebrate mosquito vector and vertebrate primate host, which have a distinct difference in their preference of codon pairs (CP) for translation (different “codon pair bias”). Consequently, arboviruses must delicately balance the use of codon pairs between mammals and arthropods, which presents an Achilles’ heel that we have exploited by specifically shifting the codon pair preference in the E and NS3 ORFs away from mammals while keeping the CPB favorable for mosquito ORFs. Here we report that recoding of the ORFs has led to variants that were over-attenuated in rhesus macaques although induction of protective antibodies in animals vaccinated with the smallest recoded ORF (E) was observed. The flexibility of our synthetic vaccine design (by decreasing the number of unfavorable CPs in the E ORF), allowed us to construct two new vaccine candidates (EhminA and EhminB) with intermediate attenuation in cell culture and neonatal mice, a result demonstrating proof of concept. New DENV vaccine candidates are being developed based on selective attenuation by dramatic recoding, with flexibility in balancing the attenuation and immunogenicity by marrying rational design and empirical modification.

The Role of Orthogonality in Genetic Code Expansion.

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

Attenuation of Human Respiratory Viruses by Synonymous Genome Recoding.

Using computer algorithms and commercial DNA synthesis, one or more ORFs of a microbial pathogen such as a virus can be recoded and deoptimized by several strategies that may involve the introduction of up to thousands of nucleotide (nt) changes without affecting amino acid (aa) coding. The synonymous recoding strategies that have been applied to RNA viruses include: deoptimization of codon or codon-pair usage, which may reduce protein expression among other effects; increased content of immunomodulatory CpG and UpA RNA, which increase immune responses and thereby restrict viral replication; and substitution of serine and leucine codons with synonymous codons for which single-nt substitutions can yield nonsense codons, thus limiting evolutionary potential. This can reduce pathogen fitness and create potential live-attenuated vaccines that may have improved properties. The combined approach of genome recoding, synthetic biology, and reverse genetics offers several advantages for the generation of attenuated RNA viruses. First, synonymous recoding involves many mutations, which should reduce the rate and magnitude of de-attenuation. Second, increasing the amount of recoding can provide increased attenuation. Third, because there are no changes at the aa level, all of the relevant epitopes should be expressed. Fourth, attenuation frequently does not compromise immunogenicity, suggesting that the recoded viruses have increased immunogenicity per infectious particle. Synonymous deoptimization approaches have been applied to two important human viral pathogens, namely respiratory syncytial virus (RSV) and influenza A virus (IAV). This manuscript will briefly review the use of these different methods of synonymous recoding to generate attenuated RSV and IAV strains. It also will review the characterization of these vaccine candidates in vitro and in animal models, and describe several surprising findings with respect to phenotypic and genetic instability of some of these candidates.