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1.
Nat Commun ; 12(1): 792, 2021 02 04.
Article in English | MEDLINE | ID: mdl-33542232

ABSTRACT

The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.


Subject(s)
Cell Engineering/methods , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Chimeric Antigen/genetics , Recombinant Fusion Proteins/genetics , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Communication/immunology , Cell Line, Tumor , Female , HEK293 Cells , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Neoplasms/immunology , Neoplasms/pathology , Primary Cell Culture , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Synthetic Biology/methods , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Xenograft Model Antitumor Assays
2.
Sci Rep ; 10(1): 3000, 2020 Feb 14.
Article in English | MEDLINE | ID: mdl-32060414

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

3.
Nat Commun ; 10(1): 4845, 2019 10 24.
Article in English | MEDLINE | ID: mdl-31649244

ABSTRACT

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.


Subject(s)
Cold Temperature , DNA/metabolism , Genetic Engineering/methods , Light , Recombinases/genetics , Animals , DNA Nucleotidyltransferases , Gene Regulatory Networks , HEK293 Cells , Humans , Integrases , Mice , Recombinases/metabolism
4.
Cell ; 173(6): 1426-1438.e11, 2018 05 31.
Article in English | MEDLINE | ID: mdl-29706540

ABSTRACT

T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical "upgrades," such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system.


Subject(s)
Lymphocyte Activation/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocyte Subsets/immunology , Animals , Antigens , Female , Humans , Immunotherapy , Jurkat Cells , K562 Cells , Mice , Mice, Inbred NOD , Neoplasm Transplantation , Neoplasms/immunology , Recombinant Fusion Proteins/immunology , Signal Transduction
5.
Sci Rep ; 8(1): 3846, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29497107

ABSTRACT

Axl is a tyrosine kinase receptor that is commonly overexpressed in many cancers. As such, Axl represents an attractive therapeutic target. The transfer of engineered T cell expressing chimeric antigen receptor (CAR) is an exciting cancer therapeutic approach that shows high efficacy against cancers in clinical trials, especially for B cell malignancies. Furthermore, recently developed synthetic Notch (synNotch) receptor has demonstrated potential in enhancing the specificity of CAR T cell therapy and delivering therapeutic payloads to tumors in an antigen-dependent manner. Therefore, a CAR or synNotch against Axl could be a valuable therapeutic reagent against many cancers. Here, we develop a single-chain variable fragment from a humanized monoclonal antibody against Axl. The scFv is attached to CD3ζ, CD28, and 4-1BB signaling domains to generate an anti-Axl CAR. When introduced into human primary T cells, the anti-Axl CAR can lead to cytokine production and cell killing in response to tumor cells expressing Axl. Moreover, an anti-Axl synNotch generated using the same scFv can be activated with Axl expressing tumor cells. Given the fact that Axl is an important cancer therapeutic target, these receptors could be valuable reagents for developing anti-Axl therapies.


Subject(s)
Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Chimeric Antigen/metabolism , Receptors, Notch/metabolism , Antibodies, Monoclonal, Humanized , Cell Line, Tumor , Humans , Immunotherapy, Adoptive , Jurkat Cells , K562 Cells , Protein Engineering/methods , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Notch/genetics , Signal Transduction , Single-Chain Antibodies/immunology , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine Kinase
6.
Annu Rev Biomed Eng ; 20: 95-118, 2018 06 04.
Article in English | MEDLINE | ID: mdl-29345976

ABSTRACT

Cellular immunotherapy holds great promise for the treatment of human disease. Clinical evidence suggests that T cell immunotherapies have the potential to combat cancers that evade traditional immunotherapy. Despite promising results, adverse effects leading to fatalities have left scientists seeking tighter control over these therapies, which is reflected in the growing body of synthetic biology literature focused on developing tightly controlled, context-independent parts. In addition, researchers are adapting these tools for other uses, such as for the treatment of autoimmune disease, HIV infection, and fungal interactions. We review this body of work and devote special attention to approaches that may lend themselves to the development of an "ideal" therapy: one that is safe, efficient, and easy to manufacture. We conclude with a look toward the future of immunotherapy: how synthetic biology can shift the paradigm from the treatment of disease to a focus on wellness and human health as a whole.


Subject(s)
Cell- and Tissue-Based Therapy , Immunotherapy/methods , Synthetic Biology , Autoimmune Diseases/therapy , HIV Infections/therapy , Humans , Immunologic Factors/therapeutic use , Mycoses/therapy , Neoplasms/therapy , Patient Safety , T-Lymphocytes/immunology
7.
BMC Syst Biol ; 11(1): 1, 2017 01 06.
Article in English | MEDLINE | ID: mdl-28061857

ABSTRACT

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored. RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally. CONCLUSIONS: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.


Subject(s)
Escherichia coli/physiology , Protein Interaction Mapping , Computational Biology , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Profiling , Hydrogen-Ion Concentration , Phenotype
8.
Nat Chem Biol ; 13(2): 131-132, 2017 01 19.
Article in English | MEDLINE | ID: mdl-28103225

Subject(s)
Synthetic Biology
9.
Integr Biol (Camb) ; 8(4): 504-17, 2016 Apr 18.
Article in English | MEDLINE | ID: mdl-27068224

ABSTRACT

Investigations into cells and their contents have provided evolving insight into the emergence of complex biological behaviors. Capitalizing on this knowledge, synthetic biology seeks to manipulate the cellular machinery towards novel purposes, extending discoveries from basic science to new applications. While these developments have demonstrated the potential of building with biological parts, the complexity of cells can pose numerous challenges. In this review, we will highlight the broad and vital role that the synthetic biology approach has played in applying fundamental biological discoveries in receptors, genetic circuits, and genome-editing systems towards translation in the fields of immunotherapy, biosensors, disease models and gene therapy. These examples are evidence of the strength of synthetic approaches, while also illustrating considerations that must be addressed when developing systems around living cells.


Subject(s)
Genetic Engineering/methods , Immunotherapy/methods , Neoplasms/genetics , Neoplasms/immunology , Synthetic Biology/methods , Animals , CRISPR-Cas Systems , Gene Editing , Gene Regulatory Networks , Genetic Therapy , Genome , Humans , Models, Biological , Oscillometry , Receptors, Antigen/chemistry
10.
IEEE Trans Med Imaging ; 34(2): 678-89, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25361500

ABSTRACT

Statistical image reconstruction methods for X-ray computed tomography (CT) provide improved spatial resolution and noise properties over conventional filtered back-projection (FBP) reconstruction, along with other potential advantages such as reduced patient dose and artifacts. Conventional regularized image reconstruction leads to spatially variant spatial resolution and noise characteristics because of interactions between the system models and the regularization. Previous regularization design methods aiming to solve such issues mostly rely on circulant approximations of the Fisher information matrix that are very inaccurate for undersampled geometries like short-scan cone-beam CT. This paper extends the regularization method proposed in to 3-D cone-beam CT by introducing a hypothetical scanning geometry that helps address the sampling properties. The proposed regularization designs were compared with the original method in with both phantom simulation and clinical reconstruction in 3-D axial X-ray CT. The proposed regularization methods yield improved spatial resolution or noise uniformity in statistical image reconstruction for short-scan axial cone-beam CT.


Subject(s)
Imaging, Three-Dimensional/methods , Models, Statistical , Tomography, X-Ray Computed/methods , Computer Simulation , Humans , Phantoms, Imaging
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