RESUMEN
Immune cell therapies are an emerging class of living drugs that rely on the delivery of therapeutic transgenes to enhance, modulate, or restore cell function, such as those that encode for tumor-targeting receptors or replacement proteins. However, many cellular immunotherapies are autologous treatments that are limited by high manufacturing costs, typical vein-to-vein time of 3-4 weeks, and severe immune-related adverse effects. To address these issues, different classes of gene delivery vehicles are being developed to target specific immune cell subsets in vivo to address the limitations of ex vivo manufacturing, modulate therapeutic responses in situ, and reduce on- and off-target toxicity. The success of in vivo gene delivery to immune cells - which is being tested at the preclinical and clinical stages of development for the treatment of cancer, infectious diseases, and autoimmunity - is paramount for the democratization of cellular immunotherapies.
RESUMEN
Genetically encoded fluorescent indicators (GEFIs) are protein-based optogenetic tools that change their fluorescence intensity when binding specific ligands in cells and tissues. GEFI encoding DNA can be expressed in cell subtypes while monitoring cellular physiological responses. However, engineering GEFIs with physiological sensitivity and pharmacological specificity often requires iterative optimization through trial-and-error mutagenesis while assessing their biophysical function in vitro one by one. Here, the vast mutational landscape of proteins constitutes a significant obstacle that slows GEFI development, particularly for sensors that rely on mammalian host systems for testing. To overcome these obstacles, we developed a multiplexed high-throughput engineering platform called the optogenetic microwell array screening system (Opto-MASS) that functionally tests thousands of GEFI variants in parallel in mammalian cells. Opto-MASS represents the next step for engineering optogenetic tools as it can screen large variant libraries orders of magnitude faster than current methods. We showcase this system by testing over 13,000 dopamine and 21,000 opioid sensor variants. We generated a new dopamine sensor, dMASS1, with a >6-fold signal increase to 100 nM dopamine exposure compared to its parent construct. Our new opioid sensor, µMASS1, has a â¼4.6-fold signal increase over its parent scaffold's response to 500 nM DAMGO. Thus, Opto-MASS can rapidly engineer new sensors while significantly shortening the optimization time for new sensors with distinct biophysical properties.