Inadequate oxygen supply, or hypoxia, is characteristic of the tumor microenvironment and correlates with poor prognosis and therapeutic resistance. Hypoxia leads to the activation of the hypoxia-inducible factor (HIF) signaling pathway and stabilization of the HIF-α subunit, driving tumor progression. The homologous alpha subunits, HIF-1α and HIF-2α, are responsible for mediating the transcription of a multitude of critical proteins that control proliferation, angiogenic signaling, metastasis, and other oncogenic factors, both differentially and sequentially regulating the hypoxic response. Post-translational modifications of HIF play a central role in its behavior as a mediator of transcription, as well as the temporal transition from HIF-1α to HIF-2α that occurs in response to chronic hypoxia. While it is evident that HIF-α is highly dynamic, HIF-2α remains vastly under-considered. HIF-2α can intensify the behaviors of the most aggressive tumors by adapting the cell to oxidative stress, thereby promoting metastasis, tissue remodeling, angiogenesis, and upregulating cancer stem cell factors. The structure, function, hypoxic response, spatiotemporal dynamics, and roles in the progression and persistence of cancer of this HIF-2α molecule and its EPAS1 gene are highlighted in this review, alongside a discussion of current therapeutics and future directions.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that lives on surfaces and skin and can cause serious infections inside the body. Antimicrobial peptides (AMPs) are part of the innate immune system and can eliminate pathogens, including bacteria and viruses, and are a promising alternative to antibiotics. Although studies have reported that AMP-functionalized hydrogels can prevent bacterial adhesion and biofilm formation, AMP dosing and the combined effects of multiple AMPs are not well understood. Here, three AMPs with different antibacterial properties were synthesized and the soluble minimum inhibitory concentrations (MICs) of each AMP against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were determined. Hydrogels with immobilized AMPs at their MIC (DD₁₃-RIP 27.5 µM; indolicidin 43.8 µM; P10 120 µM) were effective in preventing MRSA adhesion and biofilm formation. Checkerboard AMP screens identified synergy between indolicidin (3.1 µM) and P10 (12.5 µM) based on soluble fractional inhibitory concentration indices (FICIs) against MRSA, and hydrogels formed with these AMPs at half of their synergistic concentrations (total peptide concentration, 7.8 µM) were highly efficacious in killing MRSA. Mammalian cells cultured atop these hydrogels were highly viable, demonstrating that these AMP hydrogels are biocompatible and selectively eradicate bacteria, based on soluble checkerboard-screening data.

A major aim in the field of synthetic biology is developing tools capable of responding to user-defined inputs by activating therapeutically relevant cellular functions. Gene transcription and regulation in response to external stimuli are some of the most powerful and versatile of these cellular functions being explored. Motivated by the success of chimeric antigen receptor (CAR) T-cell therapies, transmembrane receptor-based platforms have been embraced for their ability to sense extracellular ligands and to subsequently activate intracellular signal transduction. The integration of transmembrane receptors with transcriptional activation platforms has not yet achieved its full potential. Transient expression of plasmid DNA is often used to explore gene regulation platforms in vitro. However, applications capable of targeting therapeutically relevant endogenous or stably integrated genes are more clinically relevant. Gene regulation may allow for engineered cells to traffic into tissues of interest and secrete functional proteins into the extracellular space or to differentiate into functional cells. Transmembrane receptors that regulate transcription have the potential to revolutionize cell therapies in a myriad of applications, including cancer treatment and regenerative medicine. In this review, we will examine current engineering approaches to control transcription in mammalian cells with an emphasis on systems that can be selectively activated in response to extracellular signals. We will also speculate on the potential therapeutic applications of these technologies and examine promising approaches to expand their capabilities and tighten the control of gene regulation in cellular therapies.

Transmembrane receptors that endow mammalian cells with the ability to sense and respond to biomaterial-bound ligands will prove instrumental in bridging the fields of synthetic biology and biomaterials. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning, and this study reports the rational design of synthetic receptors that reversibly activate cellular responses based on peptide-ligand recognition. This transmembrane receptor platform, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDAs phosphorylate a substrate that merges two protein halves, whereas Inhibitory EPDAs revert split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2000 candidate monobodies were built in silico using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time- and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and regenerative medicine.