Rapid advances in synthetic biology are driving the development of genetically engineered microbes as therapeutic agents for a multitude of human diseases, including cancer. The immunosuppressive microenvironment of solid tumors, in particular, creates a favorable niche for systemically administered bacteria to engraft and release therapeutic payloads. However, such payloads can be harmful if released outside the tumor in healthy tissues where the bacteria also engraft in smaller numbers. To address this limitation, we engineer therapeutic bacteria to be controlled by focused ultrasound, a form of energy that can be applied noninvasively to specific anatomical sites such as solid tumors. This control is provided by a temperature-actuated genetic state switch that produces lasting therapeutic output in response to briefly applied focused ultrasound hyperthermia. Using a combination of rational design and high-throughput screening we optimize the switching circuits of engineered cells and connect their activity to the release of immune checkpoint inhibitors. In a clinically relevant cancer model, ultrasound-activated therapeutic microbes successfully turn on in situ and induce a marked suppression of tumor growth. This technology provides a critical tool for the spatiotemporal targeting of potent bacterial therapeutics in a variety of biological and clinical scenarios.

Ultrasound allows imaging at a much greater depth than optical methods, but existing genetically encoded acoustic reporters for in vivo cellular imaging have been limited by poor sensitivity, specificity and in vivo expression. Here we describe two acoustic reporter genes (ARGs)—one for use in bacteria and one for use in mammalian cells—identified through a phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea that provide stronger ultrasound contrast, produce non-linear signals distinguishable from background tissue and have stable long-term expression. Compared to their first-generation counterparts, these improved bacterial and mammalian ARGs produce 9-fold and 38-fold stronger non-linear contrast, respectively. Using these new ARGs, we non-invasively imaged in situ tumor colonization and gene expression in tumor-homing therapeutic bacteria, tracked the progression of tumor gene expression and growth in a mouse model of breast cancer, and performed gene-expression-guided needle biopsies of a genetically mosaic tumor, demonstrating non-invasive access to dynamic biological processes at centimeter depth.

The Sex-determining Region Y (SRY)-related high-mobility-group box (Sox) gene family is comprised of 20 genes in vertebrates and 12 genes in invertebrates (Wegner, 2010). The Sox family of transcription factors contain the high-mobility-group (HMG) box DNA-binding domain. Though there are discrepancies, the Sox genes classification include 10 groups A to J (Wegner, 2010). The SoxD subfamily amino acid sequence does not contain transactivation or trans-repression domains. However, they have function in transcriptional activation and repression (Han & Lefebvre, 2008; Lefebvre, 2010). Detailed information about the structure, expression and regulation, biological function, and medical relevance of SoxD subfamily has been reviewed elsewhere (Lefebvre, 2010). The SoxD subfamily includes the transcription factors Sox5, Sox6, Sox13, and Sox23. Several studies demonstrated that Sox6 contains DNA binding domain and binds to the minor groove of DNA (Hagiwara, 2011; Han & Lefebvre, 2008). Furthermore, Sox6 function in gene regulation is multifaceted and as such it can function by directly binding to DNA, or interacting with cofactors, or micro-RNAs (miR

Background: Renal artery stenosis (RAStenosis) or renal artery occlusion is an intractable problem affecting about 6% of people over 65 and up to 40% of the people with coronary or peripheral vascular disease in the Unites States. In RAStenosis, the renal renin angiotensin aldosterone system (RAAS) plays a key role, with renin recognized as the disease driver. Renin is mainly produced in the kidney and in this study, we will determine a new function for the transcription factor Sox6 in the control of renal renin during RAStenosis. Method: We hypothesize that knocking out Sox6 in Ren1d positive cells will protect mice against renovascular hypertension, and kidney injury. To test our hypothesis, we used a new transgenic mouse model the Ren1dcre/Sox6fl/fl (Sox6 KO). In this mouse, Sox6 is knockout in renin expressing cells. We used a modified two kidney one clip (2K1C) Goldblatt mouse model to induce RAStenosis and renovascular hypertension. Blood pressure was measured with tail-cuff method. Renin, prorenin, Sox6, and N-GAL expressions levels were measured with Western blot, in situ hybridization, and immunohistochemistry. Creatinine levels were measured with colorimetric assay. Results: Systolic blood pressure was significantly lower in Sox6 KO two weeks after RAStenosis compared to Sox6 WT (Ren1dcre/Sox6wt/wt). When stenosed kidneys were compared, renin, prorenin, and N-GAL expressions levels in the kidney were lower in Sox6 KO compared to Sox6 WT mice. Furthermore, creatinine clearance was preserved in Sox6 KO compared to Sox6 WT mice. Conclusions: Our data indicate that Sox6 controls renal renin and prorenin expression and as such has a new function in renovascular hypertension induced by RAStenosis. These results point to a novel transcriptional regulatory network controlled by Sox6.

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