Biology subjects

Riedmayr, L. M.

Publications and source records attributed to Riedmayr, L. M..

4 recordsLinked to original sources

bioRxiv2024

Site of breast cancer metastasis is independent of single nutrient levels

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. In this study, we quantify the absolute levels of over 100 nutrients available across multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different organs. We then asked how tumor growth in different tissues relates to nutrient availability and tumor biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. Additionally, we identify purine synthesis as a requirement for tumor growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumor de novo nucleotide synthesis activity. These data suggest that a complex interplay of multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

cancer biology↗

bioRxiv2024

Machine Learning and Directed Evolution of Base Editing Enzymes

As we enter the era of CRISPR medicines, base editors (BEs) emerged as one of the most promising tools to treat genetic associated diseases. However, unintended bystander editing beyond the target nucleotide poses a challenge to their translation into effective therapies. While many efforts have been made in the design of a universal enzyme with minimal bystander editing, the context dependent activity represents a major challenge for base editing-based therapies. In this work, we designed a sequence-specific guide RNA library with 3-extensions and detected guides that were able to reduce bystander and increase editing efficiency in a context dependent manner. The best candidate was later used for phage assisted non-continuous evolution to find a new generation of precise base editors. Simultaneously, we use protein language models trained on massive protein sequence datasets to find the evolutionarily plausible mutational patterns that can improve deaminase activity and precision. Both strategies provide a collection of precise TadA variants that not only drastically reduced bystander edits, but also was not in detriment of on-target activity. Our findings introduce a guide/enzyme parallel engineering pipeline, which lays the foundation for the development of new personalized genome editing strategies, ultimately enhancing the safety and precision of this groundbreaking technology.

synthetic biology↗

bioRxiv2024

Randomizing the human genome by engineering recombination between repeat elements

While protein-coding genes are characterized increasingly well, 99% of the human genome is non-coding and poorly understood. This gap is due to a lack of tools for engineering variants that affect sequence to the necessary extent. To bridge this gap, we have developed a toolbox to create deletions, inversions, translocations, and extrachromosomal circular DNA at scale by highly multiplexed insertion of recombinase recognition sites into repetitive sequences with CRISPR prime editing. Using this strategy, we derived stable human cell lines with several thousand clonal insertions, the highest number of novel sequences inserted into single human genomes. Subsequent recombinase induction generated an average of more than one hundred megabase-sized rearrangements per cell, and thousands across the whole population. The ability to detect rearrangements as they are generated and to track their abundance over time allowed us to measure the selection pressures acting on different types of structural changes. We observed a consolidation towards shorter variants that preferentially delete growth-inhibiting genes and a depletion of translocations. We isolated and characterized 21 clones with multiple recombinase-induced rearrangements. These included viable haploid clones with deletions that span hundreds of kilobases as well as triploid HEK293T clones with aneuploidies and fold back chromosomes. We mapped the impact of these genetic changes on gene expression to decipher how structural variants affect gene regulation. The genome scrambling strategy developed here makes it possible to delete megabases of sequence, move sequences between and within chromosomes, and implant regulatory elements into new contexts which will shed light on the genome organization principles of humans and other species.

genomics↗

bioRxiv2023

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules, such as gene activators (CRISPRa), require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, intravitreal supplementation of ABCA4 via REVeRT improves retinal degeneration and function in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

bioengineering↗