Biology subjects

Budde, M. W.

Publications and source records attributed to Budde, M. W..

4 recordsLinked to original sources

bioRxiv2025

Engineered protein circuits for cancer therapy

A central challenge in cancer therapy is selectively killing cancer cells while minimizing resistance. Here, we engineer modular protease-based protein circuits that sense mutant RAS, the most frequently mutated oncogene in cancer, and conditionally activate cell death. Delivered transiently as mRNA in lipid nanoparticles (LNPs), circuits selectively eliminated RAS-mutant cancer cells in culture and suppressed aggressive, multifocal RAS-driven liver tumors. Compared to RAS inhibitors, circuits killed cancer cells independently of oncogene addiction, achieved potent cytotoxicity rather than cytostasis, and functioned at low RAS occupancy through catalysis rather than stoichiometric inhibition. Critically, circuits acquired minimal to no resistance under prolonged selection, while remaining potent against prevalent drug-resistance mechanisms including RAS amplification and bypass signaling. Together, these results establish design principles for engineering therapeutic protein circuits and highlight their potential to overcome longstanding limitations of existing therapeutic modalities.

synthetic biology↗

bioRxiv2023

Tuning methylation-dependent silencing dynamics by synthetic modulation of CpG density

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

synthetic biology↗

bioRxiv2020

Synthetic mammalian signaling circuits for robust cell population control

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed Paradaux, in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales, of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, the same cells limited population growth, but were more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system, and demonstrate that paradoxical circuit architectures can provide robust population control.

synthetic biology↗

bioRxiv2020

Imaging cell lineage with a synthetic digital recording system

Multicellular development depends on the differentiation of cells into specific fates with precise spatial organization. Lineage history plays a pivotal role in cell fate decisions, but is inaccessible in most contexts. Engineering cells to actively record lineage information in a format readable in situ would provide a spatially resolved view of lineage in diverse developmental processes. Here, we introduce a serine integrase-based recording system that allows in situ readout, and demonstrate its ability to reconstruct lineage relationships in cultured stem cells and flies. The system, termed intMEMOIR, employs an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. intMEMOIR accurately reconstructed lineage trees in stem cells and enabled simultaneous analysis of single cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable clonal analysis and recording in diverse systems. One sentence summaryA new genetic editing system termed intMEMOIR reveals the lineage histories of individual cells directly within their native tissue context.

synthetic biology↗