stability

Season 3, Episode 6: Screening for Enhanced RNA Vaccines with Kathrin Leppek, Gun Byeon, and Hannah Wayment-Steele

First Author: Kathrin Leppek, Gun Byeon, and Hannah Wayment-Steele

Episode Summary: When COVID-19 hit and society decided to use mRNA vaccines for the first time, many questions remained about whether RNA itself was ready for the challenge. But three scientists at Stanford University who had barely worked with each other before the pandemic realized that RNA’s limitations were merely a design challenge and not an issue with the substrate itself. Through emails and zooms, Kathrin, Gun, and Hannah built a tool to massively test RNA designs. With it, they screened for RNA with better functionality, increasing the stability and expression of the protein they encode and ultimately creating a platform to improve these life-saving vaccines.

About the Authors

  • Hannah, Gun, and Kathrin had all been separately researching various aspects of genetics and RNA before the pandemic.

  • When COVID hit and RNA vaccines were being built, the three realized they had newly complementary skill sets.

  • They set aside their individual projects, leveraged their unique backgrounds, and worked in shifts to abide by social distance rules in order to solve multiple issues facing RNA as a substrate for vaccines.

Key Takeaways

  • RNA holds great potential for therapies and vaccines as they are highly programmable, extremely flexible, and are much easier to scale than other options.

  • But RNA is hard to deploy for vaccines because it is extremely unstable both in the body and on the shelf.

  • Enhancing the expression and stability of RNA allows us to reduce the amount needed to give a person, increasing the number of people that can be vaccinated.

  • The three designed PERSIST-seq to test a multitude of RNA designs in one-pot by leveraging synthetic biology and next generation sequencing.

  • They also leveraged citizen science through a “game” called Eterna in order to optimize sequences using the collective brain power of humanity.

  • With it in they found synonymous mutations and alterations to the untranslated regions that changed RNA folding and improved stability and translation.

Translation

  • PERSIST-seq must still be validated in animal models to fully connect how improvements on stability and expression alter vaccine efficacy.

  • The team is ready to leverage their approach through licensing to help RNA vaccine companies improve their designs.

  • The design rules and method to discover them can be used to enhance any RNA therapeutic that will undoubtedly be coming through the pipeline soon.

Paper: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Season 3, Episode 1: New CRISPR, New Function with Leo Vo

First Author: Leo Vo

Episode Summary: In a single decade, CRISPR has made a dramatic impact on literally every facet of biotechnology. This game-changing system is traditionally programmed to make cuts at very specific parts of the genome, altering the code to cure disease. But a new class of CRISPRs discovered by Leo’s colleagues don’t simply cut DNA -- they integrate entirely new genetic material at targeted locations. With it, Leo generates a new method to perform very specific and highly efficient genome engineering on bacteria and describes the multitude of ways it can generate strains that revolutize commodity molecule synthesis and medicine.

About the Author

  • Leo is a PhD candidate who performed this work under Professor Sam Sternberg at Columbia University in New York City. Dr. Sternburg and his team are world experts in CRISPR biology having discovered multiple new CRISPR systems, including the function of Cas9 during his time in Professor Jennifer Doudna’s lab.

  • Leo was driven to become a synthetic biologist after being exposed to all the ways nature has engineered biology to overcome problems.

Key Takeaways

  • A new class of CRISPRs have been discovered that don't cut DNA but instead integrate new DNA on the genome.

  • Leo hijacks this CRISPR’s novel functionality to integrate whatever new DNA he wants into whatever location on a genome he desires.

  • Through the tools of synthetic biology, the system generates extremely targeted integrations at high efficiency in bacterial cells.

  • This CRISPR tool allows for integration of huge genetic payloads, iterative integrations, and integration of payloads at multiple locations in a single step, all of which create entirely new options for strain engineering.

  • The tool can be applied to multiple bacterial species and has proven utility in engineering the microbiome in situ as well as modifying industrially sought after strains.

Translation

  • Leo demonstrates that the system is highly effective in laboratory settings and can be optimized to overcome new challenges in new bacterial hosts.

  • The tool is undergoing further development and optimization to do population scale engineering -- making targeted and useful modifications to bacteria in communities like those seen in our gut or in nature.

  • Further research is needed to move this powerful integration tool into human cells as a novel method to overcome genetic disease and engineer future cell therapies.

Paper: CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering, Nature Biotechnology, 2020

Season 2, Episode 5: What Regulates the Regulatory T cells? with Jessica Cortez

First Author: Jessica Cortez

Episode Summary: Whether it's Multiple Sclerosis, Type 1 Diabetes, Lupus, or Crohn's Disease, autoimmunity is a rapidly growing problem that traditional pharmaceuticals have failed to completely cure. While these diseases have very different symptoms, they all have the same root cause -- the body’s immune system is attacking its own healthy organs. Lurking within ourselves are a group of T cells called regulatory T cells that have the power to suppress immune function. These cells have huge potential to be engineered and utilized as a platform to cure any autoimmune disease. Unfortunately, they easily lose their suppressive abilities and can even exacerbate autoimmunity if handled incorrectly. Looking to stabilize regulatory T cells, Jessica and her colleagues perform a CRISPR screen to map which genes are responsible for maintaining their suppressive function. Using this data, Jessica takes the first step to bring this incredibly powerful cell type to the clinic to help millions of patients suffering from a myriad of diseases.

About the Author

  • Jessica performed this work in the lab of Professor Alex Marson at the University of California, San Francisco. The Marson lab is renowned for their work in building and applying synthetic biology tools to understand and improve the therapeutic value of immune cells.

  • Jessica is driven to understand and cure autoimmune diseases because her mother, her sister, and her have all been diagnosed with autoimmune diseases.

Key Takeaways

  • Regulatory T cells can suppress immune reactions, making them an attractive therapeutic to be used to cure any autoimmune disease.

  • These regulatory T cells do not easily maintain their suppressive function, necessitating some engineering to make sure they maintain their therapeutic value.

  • With CRISPR, Jessica turned every gene off one-by-one in regulatory T cells to find which genes were involved in maintaining its suppressive function.

  • Jessica found a gene, USP22, that when expressed, inhibited regulatory T cell function making it a useful target for both autoimmunity and cancer.

Translation

  • While Jessica focused on one of the hits from the screen, there were many more that have massive potential as drug targets or as engineering steps for T cell therapies against autoimmunity.

  • Maintaining a stable regulatory T cell is the vital first step to creating a world where all autoimmune diseases are cured using cells.

Paper: CRISPR screen in regulatory T cells reveals modulators of Foxp3