To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits. 

The sound of silence: Transgene silencing in mammalian cell engineering.

The gut microbiota and its metabolites have been shown to play a pivotal role in the regulation of metabolic, endocrine and immune functions. Though the exact mechanism of action remains to be fully elucidated, available knowledge supports the ability of microbiota-fermented short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, to influence epigenetic and metabolic cascades controlling gene expression, chemotaxis, differentiation, proliferation, and apoptosis in several non-immune and immune cell subsets. While used as preferred metabolic substrates and sources of energy by colonic gut epithelial cells, most recent evidence indicates that these metabolites regulate immune functions, and in particular fine-tune T cell effector, regulatory and memory phenotypes, with direct in vivo consequences on the efficacy of chemotherapy, radiotherapy and immunotherapy. Most recent data also support the use of these metabolites over the course of T cell manufacturing, paving the way for refined adoptive T cell therapy engineering. Here, we review the most recent advances in the field, highlighting in vitro and in vivo evidence for the ability of SCFAs to shape T cell phenotypes and functions.

/pdf/microbial-short-chain-fatty-acids-a-strategy-to-tune-viuakhxx.pdf

Microbial short-chain fatty acids: a strategy to tune adoptive T cell therapy

The development of liver-based adeno-associated virus (AAV) gene therapies is facing concerns about limited efficiency and durability of transgene expression. We evaluated nonhuman primates following intravenous dosing of AAV8 and AAVrh10 vectors for over 2 years to better define the mechanism(s) of transduction that affect performance. High transduction of non-immunogenic transgenes was achieved, although expression declined over the first 90 days to reach a lower but stable steady state. More than 10% of hepatocytes contained single nuclear domains of vector DNA that persisted despite the loss of transgene expression. Greater reductions in vector DNA and RNA were observed with immunogenic transgenes. Genomic integration of vector sequences, including complex concatemeric structures, were detected in 1 out of 100 cells at broadly distributed loci that were not in proximity to genes associated with hepatocellular carcinoma. Our studies suggest that AAV-mediated transgene expression in primate hepatocytes occurs in two phases: high but short-lived expression from episomal genomes, followed by much lower but stable expression, likely from integrated vectors. 

Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration.

MHC restriction, which describes the binding of TCRs from CD4+ T cells to class II MHC proteins and TCRs from CD8+ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4+ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3β loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy. 

/pdf/a-class-mismatched-tcr-bypasses-mhc-restriction-via-an-2jgodrnb.pdf

A class-mismatched TCR bypasses MHC restriction via an unorthodox but fully functional binding geometry

Harnessing the Gut Microbiota to Potentiate the Efficacy of CAR T Cell Therapy

HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells.

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HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells.