Abstract Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.
Abstract Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.
Current limitations to on-demand drug manufacturing can be addressed by technologies that streamline manufacturing processes. Combining the production of two or more drugs into a single batch could not only be useful for research, clinical studies, and urgent therapies but also effective when combination therapies are needed or where resources are scarce. Here we propose strategies to concurrently produce multiple biologics from yeast in single batches by multiplexing strain development, cell culture, separation, and purification. We demonstrate proof-of-concept for three biologics co-production strategies: (i) inducible expression of multiple biologics and control over the ratio between biologic drugs produced together; (ii) consolidated bioprocessing; and (iii) co-expression and co-purification of a mixture of two monoclonal antibodies. We then use these basic strategies to produce drug mixtures as well as to separate drugs. These strategies offer a diverse array of options for on-demand, flexible, low-cost, and decentralized biomanufacturing applications without the need for specialized equipment.
The Natural Killer Group 2D (NKG2D) receptor plays an important role in protecting the host from infections and cancer. By recognizing ligands induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells. Because these ligands are not widely expressed on healthy adult tissue, NKG2D ligands may present a useful target for immunotherapeutic approaches in cancer. Novel therapies targeting NKG2D ligands for the treatment of cancer have shown preclinical success and are poised to enter into clinical trials. In this review, the NKG2D receptor and its ligands are discussed in the context of cancer, infection, and autoimmunity. In addition, therapies targeting NKG2D ligands in cancer are also reviewed.
NKp30 is a natural cytotoxicity receptor that is expressed on NK cells and recognizes B7-H6, which is expressed on several types of tumors but few normal cells. To target effector T cells against B7-H6+ tumors, we developed several chimeric AgRs (CARs) based on NKp30, which contain the CD28- and/or CD3ζ-signaling domains with the transmembrane domains from CD3ζ, CD28, or CD8α. The data show that chimeric NKp30-expressing T cells responded to B7-H6+ tumor cells. The NKp30 CAR-expressing T cells produced IFN-γ and killed B7-H6 ligand-expressing tumor cells; this response was dependent upon ligand expression on target cells but not on MHC expression. PBMC-derived dendritic cells also express NKp30 ligands, including immature dendritic cells, and they can stimulate NKp30 CAR-bearing T cells to produce IFN-γ, but to a lesser extent. The addition of a CD28-signaling domain significantly enhanced the activity of the NKp30 CAR in a PI3K-dependent manner. Adoptive transfer of T cells expressing a chimeric NKp30 receptor containing a CD28-signaling domain inhibited the growth of a B7-H6-expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, mice that remained tumor-free were resistant to a subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor Ags. Overall, this study demonstrates the specificity and therapeutic potential of adoptive immunotherapy with NKp30 CAR-expressing T cells against B7-H6+ tumor cells in vivo.
Chondrosarcomas represent the second most common primary bone malignancy. Despite the vulnerability of chondrosarcoma cells to nicotinamide adenine dinucleotide (NAD
