An AND-Gated Drug and Photoactivatable Cre-loxP System for Spatiotemporal Control in Cell-Based Therapeutics

Abstract

While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre­(2–59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC­(60–343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Building a Simple and Versatile Illumination System for Optogenetic Experiments

“…Once familiar with the electronics of this system, there are many ways to customize it. Additional strategies for customizing the system include placing the LED farther or closer to the sample and illuminating through filters/diffusers for homogenous illumination conditions or to prevent heating as in (Supplementary Figure 23) and Allen et al 5 . Another notable feature of our LED design is that it is encapsulated in epoxy and has a touch fastener on the back; this allows the LED to be securely placed with ease virtually anywhere: in incubators, fish tanks, animal cages, walls, etc.…”

“…Another potentially critical consideration is to prevent heating (when using the LEDs at high power) or diffusing the light for wider spread illumination. To address these considerations, the LEDs can be mounted outside of a black box with "privacy film" on the inside, as described in Supplementary Figure 23 and Allen et al 5 . Because of the simplicity of this system, taking it apart to verify, modify, upgrade, or repair modular components is not difficult.…”

Synthetic Biology Speeds Up Drug Target Discovery

“…All of these factors bring challenges to drug targets identification. A recent report created a novel system using 4-hydroxytamoxifen and blue light as external inputs, which can realize spatiotemporal control of CAR-T cell activity in solid tumor (Allen et al, 2019). Aiming at personalized medicine, it remains a puzzle that how to control the dose and time of using multidrug.…”

“…For example, DNA circuits control CAR expression and T cell activity on the inducible condition of 4-hydroxytamoxifen (Chakravarti et al, 2019). Another novel AND gate system named TamPA-Cre system, which includes inducible Magnet protein domains (nMag, pMag) and split Cre recombinase, can realize localized CAR expression by using 4-hydroxytamoxifen and blue light successively and thus control T cell activation in solid tumor (Allen et al, 2019).…”

Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches

“…Taking a slightly different approach, several studies used tethering to a cytosolic component to provide tighter regulation of a dimerization-dependent Cre recombinase enzyme. [28,37,38] The studies built on previous work showing that fusion of a steroid hormone ligand binding domain to a protein of interest (POI) can effectively sequester the POI in the cytosol through binding to HSP90. [39] The addition of hormone triggers release of the ligand binding domain from HSP90, allowing entry of the POI into the nucleus.…”