Biology subjects

Chazin-Gray, A.

Publications and source records attributed to Chazin-Gray, A..

3 recordsLinked to original sources

bioRxiv2024

Engineered Receptors for Soluble Cell-to-Cell Communication

Despite recent advances in mammalian synthetic biology, there remains a lack of modular synthetic receptors that can robustly respond to soluble ligands and in turn activate cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but to date only receptors against cell surface targets have approached clinical translation. To address this gap, we developed a receptor architecture based on synNotch, called SyNthetic Intramembrane Proteolysis Receptor (SNIPR), that has the added ability to be activated by soluble ligands, both natural and synthetic, with remarkably low baseline activity and high fold activation. SNIPRs are able to access an endocytic, pH-dependent cleavage mechanism to achieve soluble ligand sensing, in addition to employing a canonical-like pathway for detecting surface-bound ligands. We demonstrate the therapeutic capabilities of the receptor platform by localizing the activity of CAR T-cells to solid tumors where soluble disease-associated factors are expressed, bypassing the major hurdle of on-target off-tumor toxicity in bystander organs. We further applied the SNIPR platform to engineer fully synthetic signaling networks between cells orthogonal to natural signaling pathways, expanding the scope of synthetic biology. Our design framework enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

bioengineering↗

bioRxiv2023

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation and amplify signaling

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

synthetic biology↗

bioRxiv2020

Resource reallocation in engineered Escherichia coli strains with reduced genomes

A major challenge in synthetic biology is properly balancing evolved and engineered functions without compromising microbial fitness. Many microbial proteins are not required for growth in regular laboratory conditions, but it is unclear what fraction of the proteome can be eliminated to increase bioproduction and maintain fitness. Here, we investigated the effects of massive genome reduction in E. coli on the expression level and evolutionary stability of a model biosynthetic pathway to produce the pigment protodeoxyviolacein (PDV). We identified an amino acid metabolism imbalance and compromised growth that were correlated with elimination of genes associated with significant proteome fraction. Proteomic profiling suggested that increased amino acid pools are responsible for an alleviation of fitness defects associated with PDV expression. In addition, all strains with genome reductions that significantly affected the proteome exhibited decreased stability of PDV production compared to the wild-type strain under persistent PDV expression conditions despite the alleviation of fitness defects. These findings exhibit the importance of balancing evolved functions with engineered ones to achieve an optimal balance of fitness and bioproduction.

bioengineering↗