Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons.

Advances in stem cell science allow the production of different cell types in vitro either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between in vitro-derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.

Rewinding the Tape to Identify Intrinsic Determinants of Reprogramming Potential.

Via retrospective isolation of clones using Rewind, Jain et al. identified primed states of cells that reprogram to induced pluripotent stem cells. Examining clones, they find that cells retain memory of over several rounds of cell division. Moreover, they show that extrinsic factors change the number of primed cells, suggesting that there exist diverse paths of reprogramming and states of priming.

RNA-based controllers for engineering gene and cell therapies.

Engineered RNA-based genetic controllers provide compact, tunable, post-transcriptional gene regulation. As RNA devices are generally small, these devices are portable to DNA and RNA viral vectors. RNA tools have recently expanded to allow reading and editing of endogenous RNAs for profiling and programming of transcriptional states. With their expanded capabilities and highly compact, modular, and programmable nature, RNA-based controllers will support greater safety, efficacy, and performance in gene and cell-based therapies. In this review, we highlight RNA-based controllers and their potential as user-guided and autonomous systems for control of gene and cell-based therapies.

Accelerating Diverse Cell-Based Therapies Through Scalable Design.

Augmenting cells with novel, genetically encoded functions will support therapies that expand beyond natural capacity for immune surveillance and tissue regeneration. However, engineering cells at scale with transgenic cargoes remains a challenge in realizing the potential of cell-based therapies. In this review, we introduce a range of applications for engineering primary cells and stem cells for cell-based therapies. We highlight tools and advances that have launched mammalian cell engineering from bioproduction to precision editing of therapeutically relevant cells. Additionally, we examine how transgenesis methods and genetic cargo designs can be tailored for performance. Altogether, we offer a vision for accelerating the translation of innovative cell-based therapies by harnessing diverse cell types, integrating the expanding array of synthetic biology tools, and building cellular tools through advanced genome writing techniques.

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

Proliferation history and transcription factor levels drive direct conversion.

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history. By controlling for proliferation history and titrating each transcription factor, we find that conversion correlates with levels of the pioneer transcription factor Ngn2, whereas conversion shows a biphasic response to Lhx3. Increasing the proliferation rate of adult human fibroblasts generates morphologically mature, induced motor neurons at high rates. Using compact, optimized, polycistronic cassettes, we generate motor neurons that graft with the murine central nervous system, demonstrating the potential for in vivo therapies.

Supercoiling-mediated feedback rapidly couples and tunes transcription.

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation. To investigate the role of supercoiling-mediated feedback within multi-gene systems, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical inter-gene coupling. We find that gene syntax-the relative ordering and orientation of genes-defines the expression profiles, variance, burst dynamics, and inter-gene correlation of two-gene systems. Furthermore, supercoiling can enhance or weaken biochemical regulation. Our results suggest that supercoiling couples behavior between neighboring genes, providing a regulatory mechanism that tunes transcriptional variance in engineered gene networks and explains the behavior of co-localized native circuits.

Synthetic gene circuits as tools for drug discovery.

Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays.

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

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.

Evaluation of Lee et al.: Clarity and interpretation of mutual information in promoter transfer functions.

One snapshot of the peer review process for "Mapping the dynamic transfer functions of eukaryotic gene regulation" (Lee et al., 2021) appears below.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

Engineering cell fate: Applying synthetic biology to cellular reprogramming.

Cellular reprogramming drives cells from one stable identity to a new cell fate. By generating a diversity of previously inaccessible cell types from diverse genetic backgrounds, cellular reprogramming is rapidly transforming how we study disease. However, low efficiency and limited maturity have limited the adoption of in vitro-derived cellular models. To overcome these limitations and improve mechanistic understanding of cellular reprogramming, a host of synthetic biology tools have been deployed. Recent synthetic biology approaches have advanced reprogramming by tackling three significant challenges to reprogramming: delivery of reprogramming factors, epigenetic roadblocks, and latent donor identity. In addition, emerging insight from the molecular systems biology of reprogramming reveal how systems-level drivers of reprogramming can be harnessed to further advance reprogramming technologies. Furthermore, recently developed synthetic biology tools offer new modes for engineering cell fate.

Understanding and Engineering Chromatin as a Dynamical System across Length and Timescales.

Connecting the molecular structure and function of chromatin across length and timescales remains a grand challenge to understanding and engineering cellular behaviors. Across five orders of magnitude, dynamic processes constantly reshape chromatin structures, driving spaciotemporal patterns of gene expression and cell fate. Through the interplay of structure and function, the genome operates as a highly dynamic feedback control system. Recent experimental techniques have provided increasingly detailed data that revise and augment the relatively static, hierarchical view of genomic architecture with an understanding of how dynamic processes drive organization. Here, we review how novel technologies from sequencing, imaging, and synthetic biology refine our understanding of chromatin structure and function and enable chromatin engineering. Finally, we discuss opportunities to use these tools to enhance understanding of the dynamic interrelationship of chromatin structure and function.

Mitigating Antagonism between Transcription and Proliferation Allows Near-Deterministic Cellular Reprogramming.

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.

Feedback loops in biological networks.

We introduce fundamental concepts for the design of dynamics and feedback in molecular networks modeled with ordinary differential equations. We use several examples, focusing in particular on the mitogen-activated protein kinase (MAPK) pathway, to illustrate the concept that feedback loops are fundamental in determining the overall dynamic behavior of a system. Often, these loops have a structural function and unequivocally define the system behavior. We conclude with numerical simulations highlighting the potential for bistability and oscillations of the MAPK pathway re-engineered through synthetic promoters and RNA transducers to include positive and negative feedback loops.

Dynamically reshaping signaling networks to program cell fate via genetic controllers.

Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct cell processes. We demonstrate a class of genetic control systems, molecular network diverters, that interface with a native signaling pathway to route cells to divergent fates in response to environmental signals without modification of native genetic material. A method for identifying control points within natural networks is described that enables the construction of synthetic control systems that activate or attenuate native pathways to direct cell fate. We integrate opposing genetic programs by developing network architectures for reduced antagonism and demonstrate rational tuning of performance. Extension of these control strategies to mammalian systems should facilitate the engineering of complex cellular signaling systems.