Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system.

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

A vascularized model of the human liver mimics regenerative responses.

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.

Microenvironment-triggered multimodal precision diagnostics.

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 64Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer 18F-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.

Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers.

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 in vitro organoid models to building artificial tissue grafts.

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

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.

Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.

Stem cells reside in a specialized, regulatory environment termed the niche that dictates how they generate, maintain and repair tissues. We have previously documented that transplanted haematopoietic stem and progenitor cell populations localize to subdomains of bone-marrow microvessels where the chemokine CXCL12 is particularly abundant. Using a combination of high-resolution confocal microscopy and two-photon video imaging of individual haematopoietic cells in the calvarium bone marrow of living mice over time, we examine the relationship of haematopoietic stem and progenitor cells to blood vessels, osteoblasts and endosteal surface as they home and engraft in irradiated and c-Kit-receptor-deficient recipient mice. Osteoblasts were enmeshed in microvessels and relative positioning of stem/progenitor cells within this complex tissue was nonrandom and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different haematopoietic cell subsets localized to distinct locations according to the stage of differentiation. When physiological challenges drove either engraftment or expansion, bone-marrow stem/progenitor cells assumed positions in close proximity to bone and osteoblasts. Our analysis permits observing in real time, at a single cell level, processes that previously have been studied only by their long-term outcome at the organismal level.

Inhalable point-of-care urinary diagnostic platform.

Although low-dose computed tomography screening improves lung cancer survival in at-risk groups, inequality remains in lung cancer diagnosis due to limited access to and high costs of medical imaging infrastructure. We designed a needleless and imaging-free platform, termed PATROL (point-of-care aerosolizable nanosensors with tumor-responsive oligonucleotide barcodes), to reduce resource disparities for early detection of lung cancer. PATROL formulates a set of DNA-barcoded, activity-based nanosensors (ABNs) into an inhalable format. Lung cancer-associated proteases selectively cleave the ABNs, releasing synthetic DNA reporters that are eventually excreted via the urine. The urinary signatures of barcoded nanosensors are quantified within 20 min at room temperature using a multiplexable paper-based lateral flow assay. PATROL detects early-stage tumors in an autochthonous lung adenocarcinoma mouse model with high sensitivity and specificity. Tailoring the library of ABNs may enable not only the modular PATROL platform to lower the resource threshold for lung cancer early detection tools but also the rapid detection of chronic pulmonary disorders and infections.

Autonomous circadian rhythms in the human hepatocyte regulate hepatic drug metabolism and inflammatory responses.

Critical aspects of physiology and cell function exhibit self-sustained ~24-hour variations termed circadian rhythms. In the liver, circadian rhythms play fundamental roles in maintaining organ homeostasis. Here, we established and characterized an in vitro liver experimental system in which primary human hepatocytes display self-sustained oscillations. By generating gene expression profiles of these hepatocytes over time, we demonstrated that their transcriptional state is dynamic across 24 hours and identified a set of cycling genes with functions related to inflammation, drug metabolism, and energy homeostasis. We designed and tested a treatment protocol to minimize atorvastatin- and acetaminophen-induced hepatotoxicity. Last, we documented circadian-dependent induction of pro-inflammatory cytokines when triggered by LPS, IFN-ß, or Plasmodium infection in human hepatocytes. Collectively, our findings emphasize that the phase of the circadian cycle has a robust impact on the efficacy and toxicity of drugs, and we provide a test bed to study the timing and magnitude of inflammatory responses over the course of infection in human liver.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

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 traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics.

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.

Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.

Conditional Antimicrobial Peptide Therapeutics.

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.

A single-cell liver atlas of Plasmodium vivax infection.

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.

Virtual Undergraduate Research Experiences: More Than a Pandemic Stopgap.

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.

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

The utility of in vitro 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 in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo 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.

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

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 in vivo using exogenously administered nanoparticle sensors and ex vivo 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.

Cytokine signaling and hematopoietic homeostasis are disrupted in Lnk-deficient mice.

The adaptor protein Lnk, and the closely related proteins APS and SH2B, form a subfamily of SH2 domain-containing proteins implicated in growth factor, cytokine, and immunoreceptor signaling. To elucidate the physiological function of Lnk, we derived Lnk-deficient mice. Lnk(-/-) mice are viable, but display marked changes in the hematopoietic compartment, including splenomegaly and abnormal lymphoid and myeloid homeostasis. The in vitro proliferative capacity and absolute numbers of hematopoietic progenitors from Lnk(-/-) mice are greatly increased, in part due to hypersensitivity to several cytokines. Moreover, an increased synergy between stem cell factor and either interleukin (IL)-3 or IL-7 was observed in Lnk(-/-) cells. Furthermore, Lnk inactivation causes abnormal modulation of IL-3 and stem cell factor-mediated signaling pathways. Consistent with these results, we also show that Lnk is highly expressed in multipotent cells and committed precursors in the erythroid, megakaryocyte, and myeloid lineages. These data implicate Lnk as playing an important role in hematopoiesis and in the regulation of growth factor and cytokine receptor-mediated signaling.

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

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 Kras- and Trp53-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.

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

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.