100k Gene Therapy Viruses: Index #24

Researchers created 110,689 different viable AAV2 capsids, randomly adding mutations to a specific region of the protein sequence.

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As long as I have been in science, which is let's say 1960 to today, just about every five years there are major changes in technology that allow you to do things that you previously either said were just too hard or there was no way to do them — or which you hadn't imagined that you could ever do. —David Baltimore

100,000 Delivery Vehicles: AAVs—adeno-associated viruses—have a protein “shell” that wraps around the virus’s genetic material. The protein shell is called a capsid, and it can also be stuffed with DNA that encodes CRISPR machinery, for example. AAVs are used to deliver gene therapies. For a new study, published in Nature Biotechnology, researchers created 110,689 different viable AAV2 capsids, randomly adding mutations to a specific region of the protein sequence. The researchers also built a deep learning algorithm—a type of artificial neural network—and found that it could predict which DNA sequences could form AAV2 capsids that work as intended. The vast majority of AAVs that the team made were not viable; they did not assemble properly or were unable to package DNA. 58% of AAV2s with a single mutation were viable; just 0.3% of AAV2s with six mutations were viable. The work was led by a team from Google and the Wyss Institute, at Harvard and MIT.

Why It Matters: By learning the “design” rules for AAV2 vectors—in this case, by creating more than 100,000 of them and then using that data to inform a deep learning algorithm—researchers could build gene therapy systems that are less immunogenic and more specific. As scientists look to create larger AAV2 vectors, for example (which can hold more “stuff” for gene therapies), it will be valuable to know, ahead of time, which DNA sequences can actually form a viable capsid. The deep learning algorithm could save scientists a lot of time.

Original artwork for this newsletter by Davey Ho.

This graphic was made by Davey Ho. Feel free to use and adapt the illustration (with attribution) for non-commercial purposes.

Immune-Evading Vectors: More research news on AAVs (sorry). Sometimes AAVs trigger an immune response. Viruses are “foreign" and bind to a receptor called Toll-like receptor 9 (TLR9), which signals the body to produce pro-inflammatory cytokines. For a study in Science Translational Medicine, researchers at Harvard University engineered AAVs that are less able to trigger an immune response. They fused short, single-stranded DNA sequences to AAV capsids, which can then “block” TLR9. The DNA sequences, for example, could form “stem-loop structures that fit snugly into the interior of the ring structure of TLR9, preventing dimerization and activation.” In other words, those DNA sequences shut down the sentry, preventing the cell from ringing the bell and mustering the army. The team tested the new AAVs in mice, pigs and macaque monkeys. In many cases, the engineered AAVs worked as expected—no immune response. But when they injected the AAVs into the vitreous humor (the gooey bit in eyeballs) of macaque monkeys, the animals had eye inflammation.

Why It Matters: Gene therapies are becoming more common. Gene-editing techniques, delivered to target tissues, will require AAVs that are safe and effective. This paper presents a clever technique to reduce the immune response triggered by AAVs, and could probably be improved in the future.

Upgraded Artificial Cells: Some bacteria, like Rhodobacter sphaeroides, carry around chromatophores. These are small vesicles that perform photosynthesis. For a new study, published in Proceedings of the National Academy of Sciences, researchers plucked chromatophores from bacteria and dropped them into larger, artificial cells. The chromatophores could produce energy (in the form of ATP) from ADP, powering those larger cells; a bit like a quasi-mitochondria. The researchers used chromatophore-produced ATP to transcribe a gene in an artificial cell. They even used cryo-EM to study the chromatophores, and see how their ATP-producing proteins (ATP synthase, which can produce about 100 ATP per second) were arranged in physical space. This study came from researchers at the University of Bari Aldo Moro, in Italy.

Why It Matters: Bottom-up synthetic biologists want to build living cells from raw, chemical components. Most artificial cells are simple and their energy finite (or their energy is supplied externally, in the growth media). To build more complex cells, from scratch, researchers must find ways to produce energy, continuously, inside of them. This study is a step towards that larger goal.

Credit: NeonMob | Giphy

🧫 Other Studies Published This Week

Artificial Cells

  • Reconstituting natural cell elements in synthetic cells (Review). Advanced Biology. Link
  • Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells. PNAS. Open Access. Link


  • Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nature Chemical Biology. Link
  • A high-performance genetically encoded fluorescent biosensor for imaging physiological peroxynitrite. Cell Chemical Biology. Link
  • CRISPRi-mediated NIMPLY logic gate for fine-tuning the whole-cell sensing toward simple urine glucose detection. ACS Synthetic Biology. Link
  • Macrolide biosensor optimization through cellular substrate sequestration. ACS Synthetic Biology. Link
  • A reporter system for cytosolic protein aggregates in yeast. ACS Synthetic Biology. Open Access. Link
  • A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics. bioRxiv. Open Access. Link

Fundamental Discoveries

  • A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry. Nature Communications. Open Access. Link
  • Systematic characterization of mutations altering protein degradation in human cancers. Molecular Cell. Link
  • Construction of intracellular asymmetry and asymmetric division in Escherichia coli. Nature Communications. Open Access. Link
  • Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion. Biochemistry. Link
  • Intercellular communication induces glycolytic synchronization waves between individually oscillating cells. PNAS. Open Access. Link
  • A DNA-origami NanoTrap for studying the diffusion barriers formed by Phe-Gly-rich nucleoporins. bioRxiv. Open Access. Link
  • Addressing evolutionary questions with synthetic biology. OSF preprint. Open Access. Link

Genetic Circuits

  • Synthetic multistability in mammalian cells. bioRxiv. Open Access. Link
  • Winner-takes-all resource competition redirects cascading cell fate transitions. Nature Communications. Open Access. Link (Read the press release.)
  • A burden-free gene overexpression system. bioRxiv. Open Access. Link
  • Emergent oscillation induced by nutrient-modulating growth feedback. bioRxiv. Open Access. Link

Genetic Engineering & Control

  • Engineering 3D genome organization (Review). Nature Reviews Genetics. Open Access. Link
  • Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells. Biotechnology and Bioengineering. Link
  • Transcriptional control of Clostridium autoethanogenum using CRISPRi. Synthetic Biology. Open Access. Link
  • Small antisense DNA-based gene silencing enables cell-free bacteriophage manipulation and genome replication. ACS Synthetic Biology. Link
  • Programmable tools for targeted analysis of epigenetic DNA modifications (Review). Current Opinion in Chemical Biology. Open Access. Link
  • The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system. Metabolic Engineering. Link

Medicine and Diagnostics

  • Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science. Open Access. Link
  • Human heart-forming organoids recapitulate early heart and foregut development. Nature Biotechnology. Link
  • Synthetic biology in the clinic: engineering vaccines, diagnostics, and therapeutics (Review). Cell. Link

Metabolic Engineering

  • Optimized gene expression from bacterial chromosome by high-throughput integration and screening. Science Advances. Open Access. Link
  • Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli. Metabolic Engineering. Link
  • Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae. Metabolic Engineering. Link
  • Metabolic engineering of Saccharomyces cerevisiae for ethyl acetate biosynthesis. ACS Synthetic Biology. Link
  • Engineering biocatalytic solar fuel production: The PHOTOFUEL consortium (Review). Trends in Biotechnology. Link
  • Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch. Metabolic Engineering. Link
  • Successful enzyme colocalization strategies in yeast for increased synthesis of non-native products (Review). Frontiers in Bioengineering and Biotechnology. Open Access. Link
  • Lactic acid bacteria-derived α-glucans: From enzymatic synthesis to miscellaneous applications (Review). Biotechnology Advances. Link

New Technology

  • Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase. Nature Communications. Open Access. Link
  • Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nature Biotechnology. Link
  • RNA secondary structure prediction using deep learning with thermodynamic integration. Nature Communications. Open Access. Link
  • Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development. bioRxiv. Open Access. Link
  • cytoNet: Spatiotemporal network analysis of cell communities. bioRxiv. Open Access. Link


  • Towards plant resistance to viruses using protein-only RNase P. Nature Communications. Open Access. Link
  • Genome engineering for crop improvement and future agriculture (Review). Cell. Link

Protein Engineering

  • Self-assembly and regulation of protein cages from pre-organised coiled-coil modules. Nature Communications. Open Access. Link
  • Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials. Nature Communications. Open Access. Link
  • Designed folding pathway of modular coiled-coil-based proteins. Nature Communications. Open Access. Link

Systems Biology and Modelling

Model reduction of genome-scale metabolic models as a basis for targeted kinetic models. Metabolic Engineering. Open Access. Link

Until next time,