Ron Weiss

• Massachusetts Institute of Technology

Precise spatiotemporal control of gene expression and cellular differentiation is essential for engineering native-like multicellular structures. Current cell differentiation approaches typically rely on externally provided inputs whose effects are not targeted to distinct cells in the appropriate state and hence cannot spatially organize and matur...

Lipid nanoparticle (LNP)-mRNA vaccines have demonstrated protective capability in combating SARS-CoV-2. Their extensive deployment across the global population leads to the broad presence of T-cell immunity against the SARS-CoV-2 spike protein, presenting an opportunity to harness this immunological response as a universal antigen target for cancer...

In synthetic biology, genetic components are assembled to make transcriptional units, and transcriptional units are assembled into circuits to perform specific and predictable functions of a genetic device. Genetic devices have been described in bacteria, mammalian cell cultures and small organoids, yet development of programmable genetic circuits...

The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically Man5GlcNAc2 (Man-5), which normally exits the ER for further process...

Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal con...

Precisely sensing and guiding cell-state transitions via the conditional genetic activation of appropriate differentiation factors is challenging. Here we show that desired cell-state transitions can be guided via genetically encoded sensors, whereby endogenous cell-state-specific miRNAs regulate the translation of a constitutively transcribed endo...

The field of microrobotics has immensely grown in the last few decades, exhibiting several challenges as new features such as shapes, sizes, and actuation mechanisms are explored. Two of the biggest challenges faced in microrobotics are the development of a control system suited for precise microrobotic manipulation, and the ability to navigate mic...

This paper presents a control framework for magnetically actuated micron-scale robots ($\mu$bots) designed to mitigate disturbances and improve trajectory tracking. To address the challenges posed by unmodeled dynamics and environmental variability, we combine data-driven modeling with model-based control to accurately track desired trajectories us...

Micron-scale robots (μbots) have recently shown great promise for emerging medical applications. Accurate control of μbots, while critical to their successful deployment, is challenging. In this work, we consider the problem of tracking a reference trajectory using a μbot in the presence of disturbances and uncertainty. The disturbances primarily c...

Adipose stem cells (ASCs) have attracted considerable attention as potential therapeutic agents due to their ability to promote tissue regeneration. However, their limited tissue repair capability has posed a challenge in achieving optimal therapeutic outcomes. Herein, we conceive a series of lipid nanoparticles to reprogram ASCs with durable prote...

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells...

During development, cells undergo symmetry breaking into differentiated subpopulations that self-organize into complex structures.1,2,3,4,5 However, few tools exist to recapitulate these behaviors in a controllable and coupled manner.6,7,8,9 Here, we engineer a stochastic recombinase genetic switch tunable by small molecules to induce programmable...

Male infertility caused by genetic mutations is an important type of infertility. Currently, there is no reliable method in the clinic to address this medical need. The emergence of mRNA therapy provides a possible strategy for restoring mutant genes in the reproductive system. However, effective delivery of mRNA to spermatocytes remains a formidab...

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core plurip...

Micron-scale robots (ubots) have recently shown great promise for emerging medical applications, and accurate control of ubots is a critical next step to deploying them in real systems. In this work, we develop the idea of a nonlinear mismatch controller to compensate for the mismatch between the disturbed unicycle model of a rolling ubot and traje...

Regulated transgene expression is an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation, upon which most applications are based, suffers from complications such as epigenetic silencing that limit expression longevity and reliability. Constitutive transgene transcription paired w...

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial...

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated int...

Learning dynamical systems properties from data provides important insights that help us understand such systems and mitigate undesired outcomes. In this work, we propose a framework for learning spatio-temporal (ST) properties as formal logic specifications from data. We introduce SVM-STL, an extension of Signal Signal Temporal Logic (STL), capabl...

Reliable, predictable engineering of cellular behavior is one of the key goals of synthetic biology. As the field matures, biological engineers will become increasingly reliant on computer models that allow for the rapid exploration of design space prior to the more costly construction and characterization of candidate designs. The efficacy of such...

Many synthetic gene circuits are restricted to single-use applications or require iterative refinement for incorporation into complex systems. One example is the recombinase-based digitizer circuit, which has been used to improve weak or leaky biological signals. Here we present a workflow to quantitatively define digitizer performance and predict...

Building synthetic protein–based switches Synthetic circuits can potentially help to control complex biological processes, but systems based on regulating gene expression respond to stimuli at the minute to the hour time scale. Working in yeast cells, Mishra et al. report synthetic regulatory circuits based on protein phosphorylation reactions that...

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the comp...

Rewiring signaling networks imparts cells with new functionalities that are useful for engineering cell therapies and directing cell development. While much effort has gone into connecting extracellular inputs to desired outputs, less has been done to control the signal processing steps in-between. Here, we develop synthetic signal processing circu...

Adhesion-mediated cell sorting has long been considered an organizing principle in developmental biology. While most computational models have emphasized the dynamics of segregation to fully sorted structures, cell sorting can also generate a plethora of transient, incompletely sorted states. The timescale of such states in experimental systems is...

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device's output does not depend only on its intended inputs but also on its context. One contri...

Synthetic mRNA therapeutics have the potential to revolutionize healthcare as they enable patients to produce therapeutic proteins inside their own bodies. However, convenient methods that allow external control over the timing and magnitude of protein production after in vivo delivery of synthetic mRNA are lacking. In this study, we validate the i...

Therapies that synergistically stimulate immunogenic cancer cell death (ICD), inflammation and immune priming are of great interest for cancer immunotherapy. However, even multi-agent therapies often fail to trigger all of the steps necessary for self-sustaining antitumor immunity. Here we describe self-replicating RNAs encapsulated in lipid nanopa...

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect...

Regulation of transgene expression is becoming an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation upon which the majority of such applications are based suffers from complications such as epigenetic silencing, which limits the longevity and reliability of these efforts. Genet...

A significant goal of synthetic biology is to develop genetic devices for accurate and robust control of gene expression. Lack of modularity, wherein a device output does not depend uniquely on its intended inputs but also on its context, leads to poorly predictable device behavior. One contributor to lack of modularity is competition for shared li...

Microrobots have many potential uses in microbiology since they can be remotely actuated and precisely manipulated in biochemical fluids. Cellular function and response depends on biochemicals. Therefore, various delivery methods have been developed for delivering biologically relevant cargo using microrobots. However, localized targeting without p...

Biological research is relying on increasingly complex genetic systems and circuits to perform sophisticated operations in living cells. Performing these operations often requires simultaneous delivery of many genes, and optimizing the stoichiometry of these genes can yield drastic improvements in performance. However, sufficiently sampling the lar...

RNA replicons are a promising platform technology for vaccines. To evaluate the potential of lipid nanoparticle-formulated replicons for delivery of HIV immunogens, we designed and tested an alphavirus replicon expressing a self-assembling protein nanoparticle immunogen, the glycoprotein 120 (gp120) germline-targeting engineered outer domain (eOD-G...

Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs...

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the National Science Foundation (NSF), award number 1745645.

Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RN...

The development of RNA-encoded regulatory circuits relying on RNA-binding proteins (RBPs) has enhanced the applicability and prospects of post-transcriptional synthetic network for reprogramming cellular functions. However, the construction of RNA-encoded multilayer networks is still limited by the availability of composable and orthogonal regulato...

Signal Temporal Logic (STL) is a formal language for describing a broad range of real-valued, temporal properties in cyber-physical systems. While there has been extensive research on verification and control synthesis from STL requirements, there is no formal framework for comparing two STL formulae. In this paper, we show that under mild assumpti...

MicroRNAs (miRNAs) regulate a majority of protein-coding genes, affecting nearly all biological pathways. However, the quantitative dimensions of miRNA-based regulation are not fully understood. In particular, the implications of miRNA target site location, composition rules for multiple target sites, and cooperativity limits for genes regulated by...

Understanding and reshaping cellular behaviors with synthetic gene networks requires the ability to sense and respond to changes in the intracellular environment. Intracellular proteins are involved in almost all cellular processes, and thus can provide important information about changes in cellular conditions such as infections, mutations, or dis...

Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, a...

Throughout biology, function is intimately linked with form. Across scales ranging from subcellular to multiorganismal, the identity and organization of a biological structure's subunits dictate its properties. The field of molecular morphogenesis has traditionally been concerned with describing these links, decoding the molecular mechanisms that g...

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for gen...

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill fu...

Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that...

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and d...

Synthetic regulatory circuits encoded in RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We...

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its...

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce...

Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control...

Supplementary Tables

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To...

A long-standing goal of synthetic biology is to rapidly engineer new regulatory circuits from simpler devices. As circuit complexity grows, it becomes increasingly important to guide design with quantitative models, but previous efforts have been hindered by lack of predictive accuracy. To address this, we developed Empirical Quantitative Increment...

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription fact...

Described herein are methods of evaluating the expression levels of DNA parts encoding proteins in test circuits. In particular, the methods disclosed herein are useful to evaluate the expression of an output protein regulated by a regulatory protein-genetic element pair.

RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expres...

A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonst...

Noncoding small RNAs regulate gene expression through complex RNA interference (RNAi) signaling networks. The elucidation of molecular mechanisms underlying RNAi and the discovery of commonly occurring transcriptional and post-transcriptional motifs have enabled the construction of RNAi-based sensors and devices used for engineering genetic modules...

Diabetes is caused by the loss or dysfunction of insulin-secreting β-cells in the pancreas. β-cells reduce their mass and lose insulin-producing ability in vitro, likely due to insufficient cell-cell and cell-extracellular matrix (ECM) interactions as β-cells lose their native microenvironment. Herein, we built an ex-vivo cell microenvironment by c...

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up...

Pseudomonas Aeruginosa (P. aeruginosa) is a major cause of urinary tract and nosocomial infections. Here, we propose and demonstrate proof-of-principle for a potential cell therapy approach against P. aeruginosa. Using principles of synthetic biology, we genetically modified E. coli to specifically detect wild type P. aeruginosa (PAO1) via its quor...

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express a light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of a multitude of skeletal myotubes that co...

We present a workflow for the design and production of biological networks from high-level program specifications. The workflow is based on a sequence of intermediate models that incrementally translate high-level specifications into DNA samples that implement them. We identify algorithms for translating between adjacent models and implement them a...

Oscillator rate constants (see Table S1) were randomly varied across one order of magnitude around initial values (uniform distribution in the log space) to produce roughly 2000 parameter sets. Simulations of each parameter set yielded a corresponding S/N value, which is plotted here as a function of the individual parameters. Each point represents...

RS-HDMR global parametric sensitivity analysis of oscillator module rate constants (see Figure S7), describing the influence of parameter variation on observed S/N. (A) RS-HDMR first-order component functions, in order of decreasing global sensitivity index . (B) Second-order RS-HDMR component functions in order of decreasing global sensitivity ind...

Inference of the S/N values for Systems 3 and 4. (A) RS-HDMR inference of System 3 S/N value using oscillator rate constants (A) or oscillator phenotypes (B), and RS-HDMR inference of System 4 S/N value using either throttle rate constants (C) or throttle phenotypes (D). The red curve indicates the distribution of S/N observed in response to parame...

S/N values plotted against the different oscillator phenotypes (as described in Table S4) corresponding to the parameter sets of Figure S7. Multiple simulations of each parameter set yielded a phenotype in the isolated System (see Figure 8A) corresponding to the S/N value evaluated with the whole System 3. Each point represents an individual parame...