Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.

Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective T cell-mediated killing of cancer cells, but not of normal cells, in vitro. In in vivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies.

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5' UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5' UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5' UTRs. A total of ~12,000 5' UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5' UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5' UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

Item recognition and lure discrimination in younger and older adults are supported by alpha/beta desynchronization.

Our episodic memories vary in their specificity, ranging from a mere sense of familiarity to detailed recollection of the initial experience. Recent work suggests that alpha/beta desynchronization promotes information flow through the cortex, tracking the richness in detail of recovered memory representations. At the same time, as we age, memories become less vivid and detailed, which may be reflected in age-related reductions in alpha/beta desynchronization during retrieval. To understand age differences in the specificity of episodic memories, we investigated differences in alpha/beta desynchronization between younger (18-26 years, n = 31) and older (65-76 years, n = 27) adults during item recognition and lure discrimination. Alpha/beta desynchronization increased linearly with the demand for memory specificity, i.e., the requirement to retrieve details for an accurate response, across retrieval situations (correct rejections < item recognition < lure discrimination). Stronger alpha/beta desynchronization was related to memory success, as indicated by reliable activation differences between correct and incorrect memory responses. In line with the assumption of a loss of mnemonic detail in older age, older adults had more difficulties than younger adults to discriminate lures from targets. Importantly, they also showed a reduced modulation of alpha/beta desynchronization across retrieval demands. Together, these results extend previous findings by demonstrating that alpha/beta desynchronization dissociates between item recognition and the retrieval of highly detailed memories as required in lure discrimination, and that age-related impairments in episodic retrieval are accompanied by attenuated modulations in the alpha/beta band. Thus, we provide novel findings suggesting that alpha/beta desynchronization tracks mnemonic specificity and that changes in these oscillatory mechanisms may underlie age-related declines in episodic memory.

Microglia recapitulate a hematopoietic master regulator network in the aging human frontal cortex.

Microglia form the immune system of the brain. Previous studies in cell cultures and animal models suggest altered activation states and cellular senescence in the aged brain. Instead, we analyzed 3 transcriptome data sets from the postmortem frontal cortex of 381 control individuals to show that microglia gene markers assemble into a transcriptional module in a gene coexpression network. These markers predominantly represented M1 and M1/M2b activation phenotypes. Expression of genes in this module generally declines over the adult life span. This decrease was more pronounced in microglia surface receptors for microglia and/or neuron crosstalk than in markers for activation state phenotypes. In addition to these receptors for exogenous signals, microglia are controlled by brain-expressed regulatory factors. We identified a subnetwork of transcription factors, including RUNX1, IRF8, PU.1, and TAL1, which are master regulators (MRs) for the age-dependent microglia module. The causal contributions of these MRs on the microglia module were verified using publicly available ChIP-Seq data. Interactions of these key MRs were preserved in a protein-protein interaction network. Importantly, these MRs appear to be essential for regulating microglia homeostasis in the adult human frontal cortex in addition to their crucial roles in hematopoiesis and myeloid cell-fate decisions during embryogenesis.

Brain-expressed 3'UTR extensions strengthen miRNA cross-talk between ion channel/transporter encoding mRNAs.

Why protein-coding genes express transcripts with longer 3'untranslated regions (3'UTRs) in the brain rather than in other tissues remains poorly understood. Given the established role of 3'UTRs in post-transcriptional regulation of transcript abundance and their recently highlighted contributions to miRNA-mediated cross-talk between mRNAs, we hypothesized that 3'UTR lengthening enhances coordinated expression between functionally-related genes in the brain. To test this hypothesis, we annotated 3'UTRs of human brain-expressed genes and found that transcripts encoding ion channels or transporters are specifically enriched among those genes expressing their longest 3'UTR extension in this tissue. These 3'UTR extensions have high density of response elements predicted for those miRNAs that are specifically expressed in the human frontal cortex (FC). Importantly, these miRNA response elements are more frequently shared among ion channel/transporter-encoding mRNAs than expected by chance. This indicates that miRNA-mediated cross-talk accounts, at least in part, for the observed coordinated expression of ion channel/transporter genes in the adult human brain. We conclude that extension of these genes' 3'UTRs enhances the miRNA-mediated cross-talk among their transcripts which post-transcriptionally regulates their mRNAs' relative levels.

Early event-related potentials indicate context-specific target processing for eye and hand motor systems.

Concurrent eye and hand movements toward a common visual target require different motor programs based on identical visual input. We used event-related brain potentials (ERP) to determine if and when the processing of the visual target differs for the two motor systems. The N2, an index for target evaluation, was more negative for the target of a hand than of an eye movement in two experiments. A possible interpretation for this finding is different visual target processing. Targets for hand movements require a different weighting of visual information, for example concerning features such as surface structure which are important for hand but not for eye movements. In experiment 2, the early C1-component, which had an average maximum at 67 ms following target onset, was significantly more negative when subjects pointed at the stimuli. Traditionally, the C1 has been regarded as a sensory component, but recent studies have linked it to higher order processing, such as attention and expectations. Thus, the present data indicate that target processing for eye or hand movements is already context-specific during early visual information processing. We suggest that differences in a target's relevance for upcoming movements modify target processing as well as sensory expectations.