All Publications


  • Site-Programmable Transposition: Shifting the Paradigm for CRISPR-Cas Systems. Molecular cell Chavez, M. n., Qi, L. S. 2019; 75 (2): 206–8

    Abstract

    Discoveries by Klompe et al. (2019) and Strecker et al. (2019) elucidate distinct CRISPR-Cas mechanisms for site-specific programmable transposition in prokaryotic organisms.

    View details for DOI 10.1016/j.molcel.2019.07.004

    View details for PubMedID 31348878

  • Distinct immune signatures in directly treated and distant tumors result from TLR adjuvants and focal ablation THERANOSTICS Chavez, M., Silvestrini, M. T., Ingham, E. S., Fite, B. Z., Mahakian, L. M., Tam, S. M., Ilovitsh, A., Monjazeb, A. M., Murphy, W. J., Hubbard, N. E., Davis, R. R., Tepper, C. G., Borowsky, A. D., Ferrara, K. W. 2018; 8 (13): 3611–28

    Abstract

    Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments. Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components. Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation. Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

    View details for PubMedID 30026870

  • Reproducibility of High-Throughput Plate-Reader Experiments in Synthetic Biology ACS SYNTHETIC BIOLOGY Chavez, M., Ho, J., Tan, C. 2017; 6 (2): 375–80

    Abstract

    Plate-reader assays are commonly conducted to quantify the performance of synthetic biological systems. However, on the basis of a survey of 100 publications, we find that most publications do not report critical experimental settings of plate reader assays, suggesting widespread issues in their reproducibility. Specifically, critical plate reader settings, including shaking time and covering method, either vary between laboratories or are not reported by the publications. Here, we demonstrate that the settings of plate reader assays have a significant impact on bacterial growth, recombinant gene expression, and biofilm formation. Furthermore, we show that the plate reader settings affect the apparent activity, sensitivity, and chemical kinetics of synthetic constructs, as well as alter the apparent effectiveness of antibiotics. Our results suggest the critical need for consistent reporting of plate reader protocols to ensure the reproducibility of the protocols. In addition, our work provides data for the setup of plate reader protocols in synthetic biology experiments.

    View details for DOI 10.1021/acssynbio.6b00198

    View details for Web of Science ID 000394736400021

    View details for PubMedID 27797498

  • Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity. Cell reports Leylek, R. n., Alcántara-Hernández, M. n., Granja, J. M., Chavez, M. n., Perez, K. n., Diaz, O. R., Li, R. n., Satpathy, A. T., Chang, H. Y., Idoyaga, J. n. 2020; 32 (12): 108180

    Abstract

    Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.

    View details for DOI 10.1016/j.celrep.2020.108180

    View details for PubMedID 32966789

  • Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites. Proceedings of the National Academy of Sciences of the United States of America Ilovitsh, T. n., Feng, Y. n., Foiret, J. n., Kheirolomoom, A. n., Zhang, H. n., Ingham, E. S., Ilovitsh, A. n., Tumbale, S. K., Fite, B. Z., Wu, B. n., Raie, M. N., Zhang, N. n., Kare, A. J., Chavez, M. n., Qi, L. S., Pelled, G. n., Gazit, D. n., Vermesh, O. n., Steinberg, I. n., Gambhir, S. S., Ferrara, K. W. 2020

    Abstract

    Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

    View details for DOI 10.1073/pnas.1914906117

    View details for PubMedID 32430322

  • Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation. Nature communications Seo, J. W., Ingham, E. S., Mahakian, L. n., Tumbale, S. n., Wu, B. n., Aghevlian, S. n., Shams, S. n., Baikoghli, M. n., Jain, P. n., Ding, X. n., Goeden, N. n., Dobreva, T. n., Flytzanis, N. C., Chavez, M. n., Singhal, K. n., Leib, R. n., James, M. L., Segal, D. J., Cheng, R. H., Silva, E. A., Gradinaru, V. n., Ferrara, K. W. 2020; 11 (1): 2102

    Abstract

    Adeno-associated viruses (AAVs) are typically single-stranded deoxyribonucleic acid (ssDNA) encapsulated within 25-nm protein capsids. Recently, tissue-specific AAV capsids (e.g. PHP.eB) have been shown to enhance brain delivery in rodents via the LY6A receptor on brain endothelial cells. Here, we create a non-invasive positron emission tomography (PET) methodology to track viruses. To provide the sensitivity required to track AAVs injected at picomolar levels, a unique multichelator construct labeled with a positron emitter (Cu-64, t1/2 = 12.7 h) is coupled to the viral capsid. We find that brain accumulation of the PHP.eB capsid 1) exceeds that reported in any previous PET study of brain uptake of targeted therapies and 2) is correlated with optical reporter gene transduction of the brain. The PHP.eB capsid brain endothelial receptor affinity is nearly 20-fold greater than that of AAV9. The results suggest that novel PET imaging techniques can be applied to inform and optimize capsid design.

    View details for DOI 10.1038/s41467-020-15818-4

    View details for PubMedID 32355221

  • Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells. Nature communications Nakamura, M., Srinivasan, P., Chavez, M., Carter, M. A., Dominguez, A. A., La Russa, M., Lau, M. B., Abbott, T. R., Xu, X., Zhao, D., Gao, Y., Kipniss, N. H., Smolke, C. D., Bondy-Denomy, J., Qi, L. S. 2019; 10 (1): 194

    Abstract

    Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.

    View details for PubMedID 30643127

  • Reconfigurable Analog Signal Processing by Living Cells ACS SYNTHETIC BIOLOGY Lewis, D. D., Chavez, M., Chiu, K., Tan, C. 2018; 7 (1): 107–20

    Abstract

    Living cells are known for their capacity for versatile signal processing, particularly the ability to respond differently to the same stimuli using biochemical networks that integrate environmental signals and reconfigure their dynamic responses. However, the complexity of natural biological networks confounds the discovery of fundamental mechanisms behind versatile signaling. Here, we study one specific aspect of reconfigurable signal processing in which a minimal biological network integrates two signals, using one to reconfigure the network's transfer function with respect to the other, producing an emergent switch between induction and repression. In contrast to known mechanisms, the new mechanism reconfigures transfer functions through genetic networks without extensive protein-protein interactions. These results provide a novel explanation for the versatility of genetic programs, and suggest a new mechanism of signal integration that may govern flexibility and plasticity of gene expression.

    View details for DOI 10.1021/acssynbio.7b00255

    View details for Web of Science ID 000423252800013

    View details for PubMedID 29113433

  • Synthetic microbial consortia enable rapid assembly of pure translation machinery NATURE CHEMICAL BIOLOGY Villarreal, F., Contreras-Llano, L. E., Chavez, M., Ding, Y., Fan, J., Pan, T., Tan, C. 2018; 14 (1): 29-+

    Abstract

    Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

    View details for DOI 10.1038/NCHEMBIO.2514

    View details for Web of Science ID 000419964200007

    View details for PubMedID 29131146

  • Dynamic biomaterials: toward engineering autonomous feedback CURRENT OPINION IN BIOTECHNOLOGY Morris, E., Chavez, M., Tan, C. 2016; 39: 97–104

    Abstract

    Dynamic biomaterials are biocompatible engineered systems capable of sensing and actively responding to their surrounding environment. They are of growing interest, both as models in basic research to understand complex cellular systems and in medical applications. Here, we review recent advances in nano-scale and micro-scale biomaterials, specifically artificial cells consisting of compartmentalized biochemical reactions and biologically compatible hydrogels. These dynamic biomaterials respond to stimuli through triggered reactions, reaction cascades, logic gates, and autonomous feedback loops. We outline the advances and remaining challenges in implementing such 'smart' biomaterials capable of autonomously responding to environmental stimuli.

    View details for DOI 10.1016/j.copbio.2016.02.032

    View details for Web of Science ID 000378565700015

    View details for PubMedID 26974245