Tal Danino, Ph.D. - US grants

DESCRIPTION (provided by applicant): The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic bioloy. Since the original toggle switch and oscillator designs, genetic circuits have been constructed that control cellular population growth, detect edges in an image, and count discrete cellular events. One promising application of genetic circuits is to program an organism to act as an intelligent sensing and delivery device to destroy malignant cells inside the human body. Although bacterial therapies have been used to treat cancer, they have met with limited success because their inherent overgrowth and toxicity, inability to penetrate tumor environments, and specificity are challenging issues to overcome. Here, we propose to use bacterial minicells as delivery vehicles that utilize synthetic gene circuitry to selectively deliver cargo to cancer cell. Minicells are spherical nanoparticles that are produced from aberrant cell divisions in bacteria and contain no chromosome. They are able to maintain plasmids, produce energy, consume oxygen, transcribe and translate DNA, but importantly they are not able to grow and divide. Therefore, minicells can be engineered to employ intelligent decision-making by sensing the environment and delivering therapeutics in-vivo while the avoiding the efficacy issues associated with live bacterial vectors.

? DESCRIPTION (provided by applicant): Over the past decade, the long-held view of bacteria as pathogens has been transformed by microbiome data revealing an astounding prevalence of microbes within the human body. These bacteria exist not only in well- known regions such as the intestines, but also in a multitude of tissue types including tumors. Given this prevalence, microbes represent a natural platform for the development of diagnostics and therapies engineered to sense their environment and deliver drugs to tumors. Current & Mentored Research: Our preliminary results have shown that oral delivery of probiotic bacteria can colonize colorectal liver metastases and enzymatically cleave an injected substrate that can be detected in the urine (Danino et al., Science Translational Medicine, in revision). Building upon this result to improve sensitivity and specificity, we will engineer a gene circuit to lyse at a threshold population density within the tumor environment and release a urine diagnostic peptide. Circuit parameters will be predicted by mathematical modeling, characterized by microfluidic measurements, and experimentally tuned by genetic copy number and ribosome binding strengths to optimize for detection of the diagnostic peptide in mouse models. Independent Phase Research: Following initial characterization of the engineered diagnostic circuit, we will assess colonization in more realistic orthotopic and genetically engineered mouse models of colorectal cancer. We will determine earliest stages at which bacteria colonization occurs and simultaneously measure diagnostic peptide concentrations in the urine. We will target our probiotics with tumor-homing peptides, express pro- apoptotic peptides as therapeutics, and monitor progression of tumors as a function of time. Altogether, the programmable probiotic platform will allow for more sophisticated microbial diagnostics and therapeutics for cancer and establish an engineering framework for in vivo applications for synthetic biology.

Project Summary We will continue to design, construct and characterize genetic circuits. We will use micro uidic tools to grow and observe single cells and colonies in precisely controlled environmental conditions, and we will test a subset of the engineered bacterial strains as therapies in animal models. Single cell and colony dynamics will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established molecular biology techniques. Eight graduate students and postdocs will work on multiple aspects of the project, while maintaining a particular focus on modeling or technology development for monitoring bacteria or in vivo characterization. Our track record demonstrates our ability to train personnel in a multi-disciplinary approach that has led to new tools for Synthetic Biology, along with an increased understanding of gene and signaling networks generally. Our recent characterization of bacterial circuits in animal models has served to highlight the need for bene cial strains that are stable and safe over therapeutically relevant timescales. Accordingly, our Speci c Aims focus on stability (Aim 1), delivery (Aim 2), safety (Aim 3), and in vivo testing (Aim 4). Gene circuits inevitably generate mutations that are selected to decrease the additional burden created by the inserted genetic machinery. Our rst aim will develop strategies for extending the \lifetime of gene circuits in bacteria before selective pressure disables their desired functionality. We will develop computational models and experimentally quantify how circuit redundancy increases circuit lifetime. We will use our experimental platform to monitor functionality across scales from single-cell to batch culture environments. Our second aim will primarily focus on engineering small bacterial ecologies. Here we will use modeling to guide the design of up to three interacting strains that can deliver therapies in a pre-determinted sequential order. In the third aim, we will build a safety circuit that triggers the death of all bacteria at a given threshold population density. The goal is to create an irreversible intracellular switch that rapidly and eciently kills all cells before mutations can compromise the safety strategy. In the nal aim, we will test the circuits designed in the rst three aims in animal models. We will engineer optical markers that enable characterization of the dynamics of bacterial colonies and tumor size in vivo. Importantly, the relative ease and low cost of bacterial cloning will inevitably lead to a bottleneck for the eld of Synthetic Biology, as therapeutic strains can be created at a rate that will far exceed the ability to test them. This highlights an acute need for quantitative models that have been thoroughly validated using in vitro technologies. Consequently, only a fraction of the circuits built in Aims 1-3 will be deemed worthy of in vivo testing. More generally, we anticipate that the computational models arising from these studies will be generally applicable across a wide range of emerging applications that employ bacteria.

PROJECT SUMMARY/ABSTRACT In recent years, the field of cancer immunotherapy has seen a renaissance ? with the use of monoclonal antibodies that target immune checkpoints to activate anticancer immune responses demonstrating unparalleled clinical success. Several of these therapies have now gained FDA approval and are part of routine treatment regimens for several malignancies. Despite the overall success of immunotherapeutic regimens, existing modalities present complications that current research efforts seek to overcome: (1) systemic delivery of checkpoint blockade monoclonal antibodies lead to diverse and unpredictable immune-related adverse events (irAE), (2) boosting responses from the endogenous antitumor repertoire often relies on the existence of pre- primed antitumor T cells, which in the case of highly immunosuppressive tumors or those with low mutational burden may be extremely rare, and (3) attempts to combine immunotherapies to additively boost T cell responses demonstrate increased on-target, off-tumor toxicity. Thus, to circumvent toxicity and immunosuppression, contemporary strategies focus on developing methods to deliver potent immunostimulants directly into a tumor, locally priming antitumor T cells to attack disseminated metastases exhibiting a similar antigenic profile. Bridging these observations, the goals of this proposal are to engineer probiotic strains of bacteria that selectively colonize colorectal cancer (CRC) and locally release immune checkpoint blockade. We hypothesize that this approach will result in more robust and diversified antitumor T cell immunity and promote the clearance of colonized primary and metastatic colorectal cancer lesions and systemically growing CRC-derived foci. The primary innovations of this proposal are in engineering probiotics as an immunotherapeutic delivery vector to locally release high-dose immune checkpoint blockade. Specifically, the proposed system has several advantages over current therapeutic strategies, including: (1) tumor-specific production of immunotherapies and LPS adjuvant, (2) bacteria lysis, leading to effective release of novel immunotherapeutics and LPS, (3) local delivery of novel immunotherapeutics that are toxic to deliver systemically, and (4) oral delivery of probiotics that selectively colonize CRC metastases. This work seeks to shift current research and clinical practice paradigms to overcome current limitations of immunotherapies, by providing a unique vehicle to locally deliver immunotherapies that stimulate antitumor immunity while preventing systemic toxicity and mitigating irAE.

The engineering of living cells and microbes is driving a new era of cancer therapy. This transformative approach allows for the genetic programming of living cells to intelligently sense and respond to environments, ultimately adding specificity and efficacy that is otherwise unattainable with molecular-based therapeutics. Due to recent microbiome studies indicating the prevalence of bacteria within the human body and specifically in tumor tissue, bacteria have generated significant interest as cancer therapies. Additionally, a multitude of empirical studies have demonstrated that administered bacteria home and selectively grow in tumors due to reduced immune surveillance of tumor cores. Given their presence and selectivity for tumors, bacteria present a unique oppor- tunity to be engineered as intelligent delivery vehicles for cancer therapy. The objective of this proposal is to engineer and optimize S. typhimurium for metastatic colorectal cancer therapy. Since animal based-testing regimes limit the rate of clinical progress, we will use a high-throughput, bacteria-spheroid platform to rapidly test therapeutic payloads and production and release strategies. We will also assess the effect of therapies on colorectal genetic backgrounds, and investigate spatio-temporal hetero- geneity in 3D spheroids with the use of engineered cell reporters. We will then test lead candidates in mouse models of primary and metastatic colorectal cancer to evaluate safety and efficacy. We will focus on colorectal cancer due to several proof-of-concept studies from our lab demonstrating efficacy in colorectal spheroids and animal models. In particular, we showed that oral delivery of bacteria can specifically colonize colorectal liver metastases, providing an attractive delivery route as a cancer therapy. Since these metastases are often con- fined to the liver, this approach can have a significant impact on tumor growth and survival. The research in this proposal will help to establish a framework to genetically engineer microbes for cancer therapy, and significantly accelerates tools that will impact the broader cancer and synthetic biology communities. If successful, future lead candidates for potential clinical trials will be identified on the basis of therapeutic efficacy and safety studies from this proposal.

PROJECT SUMMARY/ABSTRACT Recent advances in cancer immunotherapy have provided promising treatment options for patients with triple- negative breast cancer (TNBC). Despite overall success in treating these malignancies, immunotherapeutic ap- proaches face a number of unique challenges: (1) dose limitation due to off-target side effects, (2) additive toxicity of combination therapies, (3) and relatively low immunogenicity of breast cancer. To overcome these limitations, this proposal seeks to engineer probiotic strains of bacteria that selectively colonize breast cancer and locally release immunotherapeutics. The ultimate goal is to elicit more robust and diversified antitumor T cell immunity and promote the clearance of colonized primary and metastatic breast cancer lesions and systemically growing breast cancer-derived foci. The accompanying project will first focus on deciphering mechanisms that define the intratumoral tropism of the probiotic strain E. coli Nissle 1917 (EcN) by using antibody-mediated depletion ap- proaches and targeted genetic knockouts to pinpoint host immunological pathways that regulate tumor-specific growth. Using synthetic biology approaches, EcN will then be engineered to stably express and release check- point inhibitor nanobodies targeting CD47, PD-L1, and CTLA-4 locally inside of tumors. Pro-inflammatory cyto- kines will additionally be expressed to promote antigen presentation and enhance cytotoxic T cell responses. The primary innovations of this proposal are in the combined approach of both developing a better understanding of probiotic colonization of tumors, along with engineering probiotics as an immunotherapeutic delivery vector. Specifically, this approach has several advantages over current therapeutic strategies, including: (1) identifica- tion of novel EcN host strains and mechanistic understanding of their tumor colonization for further improvements in engineered therapies, (2) tumor-specific production of immunotherapeutics, (3) bacteria lysis that leads to effective release of novel immunotherapeutics and lipopolysaccharides (LPS) adjuvant, and (4) local delivery of novel immunotherapeutic combinations that are toxic to deliver systemically. This work seeks to overcome cur- rent limitations of immunotherapies, by providing a targeted vehicle to locally deliver immunotherapies that stim- ulate antitumor immunity while preventing systemic toxicity and mitigating immune-related adverse effects.

PROJECT SUMMARY The engineering of immune cells such as chimeric antigen receptor (CAR)-T cells is driving a new era of cancer therapy. What makes this approach transformative is the ability to genetically program living cells to intelligently sense and respond to environments, adding specificity and efficacy that is not possible to obtain with molecular and antibody-based therapeutics. While CAR-T cell therapy has demonstrated remarkable success for hemato- logical malignancies, it has been faced with many challenges in solid tumor treatment including limitations in targeting safe tumor antigens, poor infiltration into tumors, and increased T cell dysfunction in the suppressive tumor microenvironment. Thus, there is a pressing need to develop technologies to enhance the safety and efficacy of this promising approach for solid tumors. Over the last few decades, microbiome and mechanistic studies have elucidated that bacteria selectively colonize tumor necrotic cores due to reduced immune surveil- lance. Where CAR-T cells struggle to target, locate, and infiltrate solid tumors, bacteria naturally home to, colo- nize, and remain indifferent to the antigenic profile of tumors. Due to advances in engineering capabilities from synthetic biology, microbes represent a natural platform for development as 'smart? therapeutic delivery vehicles for cancer. In this way, probiotics can infiltrate tumors and be engineered as beacons for directing and enhancing CAR-T cell activities. This project proposal seeks to engineer a bridge between these two complimentary forms of cellular therapies for the treatment of triple negative breast cancer (TNBC). The objective of this proposal is to engineer bacteria and CAR-T cells together, developing a ProCAR (probiotic guided CAR-T cell) system that will improve the limitation of individual agents. Specifically, probiotics will be engineered to home to tumors and locally produce immune-stimulants to enhance CAR-T cell therapies ? while CAR-T cells will be engineered to sense these molecules. The overarching innovation of this proposal is engineering communities of living medi- cines, where tumor colonizing bacteria are reengineered as beacons for directing and enhancing CAR-T cell cytotoxicity. This will be a fundamentally new approach to genetically engineering interactions between living medicines, combining the advantages of CAR-T cells and tumor-specific bacteria for cancer therapy, and further building the foundation for engineered communities.