crispr-cas-mediated-multianalyte-synthetic-urine-biomarker-test-for

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Massachusetts Institute of Technology

Howard Hughes Medical Institute

The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage the activity of the guide RNA called "CRISPR-plus" (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR-plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.

Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors.

Control of both tissue architecture and scale is a fundamental translational roadblock in tissue engineering. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. We fabricated a structurally organized engineered tissue unit that expanded in response to regenerative cues after implantation into mice with liver injury. Specifically, we found that tissues containing patterned human primary hepatocytes, endothelial cells, and stromal cells in a degradable hydrogel expanded more than 50-fold over the course of 11 weeks in mice with injured livers. There was a concomitant increase in graft function as indicated by the production of multiple human liver proteins. Histologically, we observed the emergence of characteristic liver stereotypical microstructures mediated by coordinated growth of hepatocytes in close juxtaposition with a perfused vasculature. We demonstrated the utility of this system for probing the impact of multicellular geometric architecture on tissue expansion in response to liver injury. This approach is a hybrid strategy that harnesses both biology and engineering to more efficiently deploy a limited cell mass after implantation.

In situ expansion of engineered human liver tissue in a mouse model of chronic liver disease.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

Respiratory tract infections represent a significant public health risk, and timely and accurate detection of bacterial infections facilitates rapid therapeutic intervention. Furthermore, monitoring the progression of infections after intervention enables 'course correction' in cases where initial treatments are ineffective, avoiding unnecessary drug dosing that can contribute to antibiotic resistance. However, current diagnostic and monitoring techniques rely on non-specific or slow readouts, such as radiographic imaging and sputum cultures, which fail to specifically identify bacterial infections and take several days to identify optimal antibiotic treatments.

Protease activity sensors noninvasively classify bacterial infections and antibiotic responses.

The liver plays a central role in metabolism; however, xenobiotic metabolism variations between human hepatocytes and those in model organisms create challenges in establishing functional test beds to detect the potential drug toxicity and efficacy of candidate small molecules. In the emerging areas of RNA interference, viral gene therapy, and genome editing, more robust, long-lasting, and predictive human liver models may accelerate progress. Here, we apply a new modality to a previously established, functionally stable, multi-well bioengineered microliver-fabricated from primary human hepatocytes and supportive stromal cells-in order to advance both small molecule and nucleic acid therapeutic pipelines. Specifically, we achieve robust and durable gene silencing in vitro to tune the human metabolism of small molecules, and demonstrate its capacity to query the potential efficacy and/or toxicity of candidate therapeutics. Additionally, we apply this engineered platform to test siRNAs designed to target hepatocytes and impact human liver genetic and infectious diseases.

Improving Drug Discovery by Nucleic Acid Delivery in Engineered Human Microlivers.

There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.

Lung cancer is the leading cause of cancer-related death, and patients most commonly present with incurable advanced-stage disease. U.S. national guidelines recommend screening for high-risk patients with low-dose computed tomography, but this approach has limitations including high false-positive rates. Activity-based nanosensors can detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of activity-based nanosensors for lung cancer by coupling nanosensor multiplexing with intrapulmonary delivery and machine learning to detect localized disease in two immunocompetent genetically engineered mouse models. The design of our multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma datasets and in vitro cleavage assays with recombinant candidate proteases. Intrapulmonary administration of the nanosensors to a - and -mutant lung adenocarcinoma mouse model confirmed the role of metalloproteases in lung cancer and enabled accurate detection of localized disease, with 100% specificity and 81% sensitivity. Furthermore, this approach generalized to an alternative autochthonous model of lung adenocarcinoma, where it detected cancer with 100% specificity and 95% sensitivity and was not confounded by lipopolysaccharide-driven lung inflammation. These results encourage the clinical development of activity-based nanosensors for the detection of lung cancer.

Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling.

Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic function. Engineering spatiotemporal control of cell-cell and cell-matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (Controlled Apoptosis in Multicellular tissues for Engineered Organogenesis) enables the non-invasive triggering of controlled apoptosis to eliminate genetically-engineered cells from a pre-established culture. Using this approach, the contribution of stromal cells to the phenotypic stability of primary human hepatocytes is examined. 3D hepatic microtissues, in which fibroblasts can enhance phenotypic stability and accelerate aggregation into spheroids, were found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Due to its modularity, CAMEO has the promise to be readily extendable to other applications that are tied to the complexity of 3D tissue biology, from understanding  organoid models to building artificial tissue grafts.

Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers.

Therapeutic outcomes in oncology may be aided by precision diagnostics that offer early detection, localization and the opportunity to monitor response to therapy. Here, we report a multimodal nanosensor engineered to target tumours through acidosis, respond to proteases in the microenvironment to release urinary reporters and (optionally) carry positron emission tomography probes to enable localization of primary and metastatic cancers in mouse models of colorectal cancer. We present a paradigm wherein this multimodal sensor can be employed longitudinally to assess burden of disease non-invasively, including tumour progression and response to chemotherapy. Specifically, we showed that acidosis-mediated tumour insertion enhanced on-target release of matrix metalloproteinase-responsive reporters in urine. Subsequent on-demand loading of the radiotracer Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer F-fluorodeoxyglucose. Through tailored target specificities, this modular platform has the capacity to be engineered as a pan-cancer test that may guide treatment decisions for numerous tumour types.

Microenvironment-triggered multimodal precision diagnostics.

The utility of  human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function . Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes , and also  on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.

Identification of NQO2 As a Protein Target in Small Molecule Modulation of Hepatocellular Function.

The integration of nanotechnology and synthetic biology could lay the framework for new classes of engineered biosensors that produce amplified readouts of disease states. As a proof-of-concept demonstration of this vision, here we present an engineered gene circuit that, in response to cancer-associated transcriptional deregulation, expresses heterologous enzyme biomarkers whose activity can be measured by nanoparticle sensors that generate amplified detection readouts. Specifically, we designed an AND-gate gene circuit that integrates the activity of two ovarian cancer-specific synthetic promoters to drive the expression of a heterologous protein output, secreted Tobacco Etch Virus (TEV) protease, exclusively from within tumor cells. Nanoparticle probes were engineered to carry a TEV-specific peptide substrate in order to measure the activity of the circuit-generated enzyme to yield amplified detection signals measurable in the urine or blood. We applied our integrated sense-and-respond system in a mouse model of disseminated ovarian cancer, where we demonstrated measurement of circuit-specific TEV protease activity both  using exogenously administered nanoparticle sensors and  using quenched fluorescent probes. We envision that this work will lay the foundation for how synthetic biology and nanotechnology can be meaningfully integrated to achieve next-generation engineered biosensors.

Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection.

During the SARS-CoV-2 pandemic, experimental research groups face a unique challenge: how to train undergraduates without access to labs. We share our experience developing entirely virtual undergraduate research internships and make a case for virtual research as a complement to traditional undergraduate mentoring, even after the resolution of the pandemic.

Virtual Undergraduate Research Experiences: More Than a Pandemic Stopgap.

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission.

A single-cell liver atlas of Plasmodium vivax infection.

Liver regeneration is a well-orchestrated process that is typically studied in animal models. Although previous animal studies have offered many insights into liver regeneration, human biology is less well understood. To this end, we developed a three-dimensional (3D) platform called structurally vascularized hepatic ensembles for analyzing regeneration (SHEAR) to model multiple aspects of human liver regeneration. SHEAR enables control over hemodynamic alterations to mimic those that occur during liver injury and regeneration and supports the administration of biochemical inputs such as cytokines and paracrine interactions with endothelial cells. We found that exposing the endothelium-lined channel to fluid flow led to increased secretion of regeneration-associated factors. Stimulation with relevant cytokines not only amplified the secretory response, but also induced cell-cycle entry of primary human hepatocytes (PHHs) embedded within the device. Further, we identified endothelial-derived mediators that are sufficient to initiate proliferation of PHHs in this context. Collectively, the data presented here underscore the importance of multicellular models that can recapitulate high-level tissue functions and demonstrate that the SHEAR device can be used to discover and validate conditions that promote human liver regeneration.

A vascularized model of the human liver mimics regenerative responses.

Antimicrobial peptides (AMPs) constitute a promising class of alternatives to antibiotics to curb antimicrobial resistance. Nonetheless, their utility as a systemic agent is hampered by short circulation time and toxicity. Infection sites, analogous to tumors, harbor an aberrant microenvironment that has the potential to be exploited to develop conditionally activated therapeutics with an improved therapeutic index. In particular, we identified strategies to prolong systemic circulation of small, cationic AMPs in a mouse model of bacterial pneumonia. Specifically, we report an albumin-binding domain (ABD)-AMP conjugate as a long-circulating conditional AMP therapeutic with a masked activity that can be liberated by proteases in the infected tissue microenvironment. Our systemically administered conjugate enhanced the pulmonary delivery of active AMP while also reducing AMP exposure to other off-target organs. Importantly, this reduction in off-target exposure improved the safety profile of the AMP. The framework we present can be generalized to quantify and optimize the performance of this emerging class of conditional therapeutics.

Conditional Antimicrobial Peptide Therapeutics.

Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions.

Article	Year
Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nature protocols\| PubMed ID: 26584444	2015
Development of Light-Activated CRISPR Using Guide RNAs with Photocleavable Protectors. Angewandte Chemie (International ed. in English)\| PubMed ID: 27554600	2016
In situ expansion of engineered human liver tissue in a mouse model of chronic liver disease. Science translational medicine\| PubMed ID: 28724577	2017
In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites. Cell host & microbe\| PubMed ID: 29478773	2018
Protease activity sensors noninvasively classify bacterial infections and antibiotic responses. EBioMedicine\| PubMed ID: 30503861	2018
Improving Drug Discovery by Nucleic Acid Delivery in Engineered Human Microlivers. Cell metabolism\| PubMed ID: 30840913	2019
Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes. Nanoscale\| PubMed ID: 31670340	2019
Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling. Science translational medicine\| PubMed ID: 32238573	2020
Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers. Advanced functional materials\| PubMed ID: 33776613	2020
Microenvironment-triggered multimodal precision diagnostics. Nature materials\| PubMed ID: 34267368	2021
Identification of NQO2 As a Protein Target in Small Molecule Modulation of Hepatocellular Function. ACS chemical biology\| PubMed ID: 34427427	2021
Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection. ACS synthetic biology\| PubMed ID: 34464083	2021
Virtual Undergraduate Research Experiences: More Than a Pandemic Stopgap. Med (New York, N.Y.)\| PubMed ID: 35187514	2021
A single-cell liver atlas of Plasmodium vivax infection. Cell host & microbe\| PubMed ID: 35443155	2022
A vascularized model of the human liver mimics regenerative responses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 35763565	2022
Conditional Antimicrobial Peptide Therapeutics. ACS nano\| PubMed ID: 35980829	2022
CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics. Nature nanotechnology\| PubMed ID: 37095220	2023

Heather E. Fleming

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