Genetic firewall for virus-resistance and biocontainment

The universal nature of the genetic code allows organisms to exchange functions through horizontal gene transfer (HGT) and enables recombinant gene expression in heterologous hosts. However, the shared language of the same code permits the undesired spread of antibiotic-, herbicide- and pesticide-resistance genes and allows viruses to cause diseases. 

In my first postdoctoral project, I developed a technology that renders cells resistant to natural viruses and biocontains cells and their genetic information by establishing a genetic-code-based firewall. I first discovered that viruses and horizontally transferrable mobile genetic elements easily overcome the genetic isolation of organisms that rely on a compressed genetic code (i.e., a fewer-than-64 codon genetic code), despite the lack of essential host tRNAs and Release Factor 1 necessary for viral translation. Next, I noticed that modified viral tRNAs provide exceptionally efficient codon reassignment, allowing me to establish a genetic firewall and prevent both viral infections and the escape of genetic information. 

To establish a genetic firewall, I have developed an artificial, amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation and a third codon to a non-natural amino acid (L-4,4'-biphenylalanine) to biocontain the host. 

This amino-acid-swapped genetic code renders Escherichia coli cells resistant to viral infections (including bacteriophages in environmental samples and sewage) by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. This work suggests a general strategy to make organisms safely resistant to all natural viruses and prevent genetic information flow into and out of Genetically Modified Organisms (GMOs).

This work was selected as one of the most important discoveries at Harvard Medical School in 2023, based on Harvard Medicine News.

Relevant publication & patent applications: 

• A swapped genetic code prevents viral infections and gene transfer, Akos Nyerges ✉ , Vinke S, Flynn R, Owen SV, Rand EA, Budnik B, Keen E, Narasimhan K, Marchand JA, Baas-Thomas M, Liu M, Chen K, Chiappino-Pepe A, Hu F, Baym M, Church GM ✉  (2023) Nature 2023:1–8. https://doi.org/10.1038/s41586-023-05824-z. (Jul. 8, 2022. BioRxiv) 

• Nyerges AJ, Church G. M, (2022) Methods and compositions for conferring cellular resistance to viral infection, PCT/US2023/069468

• Nyerges AJ, Church G. M, (2021) METHODS FOR MAKING AND USING GENOMICALLY RECODED CELLS, PCT/US2021/062177

Highlights:   

Nature Biotechnology; NewScientist; Synthetic Biology; Science; DOE; Nature News and Views

Interviews in Nature Podcast; Drug Discovery News; TheScientist; Harvard 

During my PhD, I developed a method that enables the precise mutagenesis of multiple, long genomic segments in multiple species without off-target modifications. This technology (DIvERGE) enables the exploration of vast numbers of combinatorial genetic alterations in their native genomic context and allows accelerated directed evolution.

I hypothesized and demonstrated that a genome engineering system I developed earlier during my undergraduate research (pORTMAGE; Nyerges, A. et al., PNAS, 2016 PMID: 26884157) enables the translation of chemical DNA-synthesis-based mutagenesis into focused genome-diversification with up to >1,000,000× the wild-type mutation rate. During my Ph.D., I scaled up this method to generate billions of combinatorial variants of multiple drug targets simultaneously and quickly discover drug-resistance processes.

Using the combinatorial mutational scanning of drug targets with DIvERGE and long-read sequencing, we identified previously undetected resistance processes for multiple antibiotics, including a clinical-stage antibiotic, gepotidacin. In a follow-up paper, we validated the discovered gepotidacin resistance mechanism’s significance in in vivo infection models (Szili, P. ..., Nyerges, A✉, Antimicrobial Agents and Chemotherapy, 2019, PMID: 31235632). Strikingly, gepotidacin’s clinical trial later revealed the same resistance process in patients. 

In connected papers, we utilized DIvERGE to perform accelerated directed evolution in multiple bacterial species, identify antibiotic resistance-conferring mutations to antibiotics in days, evolve nanobodies with affinity to novel targets, discover bacteriophage mutants to target important bacterial pathogens, and rapidly optimize genes, genetic circuits, up to entire synthetic genomes.

This technology was later outlicensed for drug development.

Relevant publications & Patent: 

• Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance, Akos Nyerges ✉, Csörgő, B., Draskovits, G., Kintses, B., Szili, P., Ferenc, G., Révész, T., Ari, E., Nagy, I., Bálint, B., Vásárhelyi, B.M., Bihari, P., Számel, M., Balogh, D., Papp, H., Kalapis, D., Papp, B., Pál, C.✉, 2018. PNAS 115, E5726–E5735. https://doi.org/10.1073/pnas.1801646115

• US10669537B2: Mutagenizing intracellular nucleic acids (Granted U.S. Patent)

• A highly precise and portable genome engineering method allows comparison of mutational effects, Akos Nyerges*, Csörgő, B*, Nagy, I., Bálint, B., Bihari, P., Lázár, V., Apjok, G., Umenhoffer, K., Bogos, B., Pósfai, G., Pál, C., 2016. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. PNAS 201520040. https://doi.org/10.1073/pnas.1520040113

Highlights:  PNAS’ In This Issue, 115(25):6315–6317.

Highlighted in “Breakthrough method predicts resistance to antibiotics under development”

In my second PhD project - in collaboration with Lucija Peterlin Mašič's team - using structure-guided rational drug design, we developed a series of novel DNA gyrase and topoisomerase IV dual-targeting antibiotics.

In this project, we have combined rational, target-based drug development with evolutionary analysis and the high-throughput prediction of resistance processes to identify key residues for drug-target interaction and suppress the evolution of drug resistance by rationally modifying our drug candidate. Next, I performed detailed preclinical testing for these compounds to demonstrate their safety and efficacy. This novel antibiotic displays broad activity against both drug-susceptible and multidrug-resistant Gram-positive bacterial pathogens, lacks toxicity in preclinical tests, was well-tolerated in mice, and demonstrated exceptional potency in mouse infection models.  

Relevant publications & Patent application: 

• Rapid evolution of reduced susceptibility against a balanced dual-targeting antibiotic through stepping-stone mutations, Szili P, Draskovits G, Révész T, Bogár F, Balogh D, Martinek T, Daruka L, Spohn R, Vásárhelyi B M, Czikkely M, Kintses B, Grézal G, Ferenc G, Pál C✉, Akos Nyerges ✉, (2019) Antimicrobial Agents and Chemotherapy, 63:10.1128/aac.00207-19. https://journals.asm.org/doi/10.1128/aac.00207-19

• PCT/EP2019/073412: New Class of DNA Gyrase and/or Topoisomerase IV Inhibitors with Activity Against Gram-Positive and Gram-Negative Bacteria

• Rational design of balanced dual-targeting antibiotics with limited resistance,  Akos Nyerges*, Tomasic T*, Durcik M*, Revesz T, Szili P, Draskovits G, Bogar F, Skok, Ž., Zidar, N., Ilaš, J., Zega, A., Kikelj, D., Daruka, L., Kintses, B., Vasarhelyi, B., Foldesi, I., Kata, D., Welin, M., Kimbung, R., Focht, D., Mašič, L.P✉, Pal C✉, (2020) PLOS Biology 18, e3000819. https://doi.org/10.1371/journal.pbio.3000819