Ready, set, then go: Rapid posttranslational induction of protein production by drug administration in vivo

ABSTRACT The ability to rapidly induce fully active proteins of interest (POIs) upon drug administration anywhere in the body would be widely useful for developmental biology, synthetic biology, and gene and cell therapies, but existing drug-regulated protein activation methods have limitations in speed, dynamic range, generalizability across proteins, and applicability across tissues. The overall goal of this proposal is to overcome these limitations by developing a novel approach, named Split Protein Ligation Activated by Steroid Hormone (SPLASH), focusing on achieving lower background, faster induction, and plug-and-play generalizability for diverse POIs. The innovative design of SPLASH involves sequestering an intein-extein fusion with the T2 mutant of the estrogen receptor hormone-binding domain (ERT2), so that 4-hydroxytamoxifen (4-OHT) can release it to trans- spice with a complementing intein-extein fusion, resulting in the production of any POI. As an example, SPLASH will be applied to improve the Cre recombinase, addressing issues of leakiness and low induced activity. This work will leverage recent atomic-level understsanding of the steps in steroid hormone receptor sequestration and release by heat-shock complexes, and recent advances in developing fast trans-splicing inteins with broad extein sequence-specificity. SPLASH will be the first method to use a steroid hormone to regulate trans-splicing, and will also introduce a transformative application for fast-splicing inteins. SPLASH will be rationally generated and optimized in two Aims. Aim 1 will focus on generating a functional SPLASH prototype by optimizing topologies, HBD-intein linkages, and intein split sites through bacterial-free library construction and expression. Aim 2 will develop SPLASH-Cre to demonstrate SPLASH utility, evaluating performance in vitro and in vivo using a bioluminescent Cre reporter, and comparing it with untagged Cre and the existing Cre-ERT2. Successful completion of this project will establish SPLASH as a robust method for creating proteins rapidly upon drug administration, providing a versatile tool for basic research, synthetic biology, and gene and cell therapies. The proposal aligns with the R21 focus on technology development, utilizing interdisciplinary approaches and state-of-the-art knowledge to address a crucial need in biomedical research.