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Biology subjects

Weiss, R.

Publications and source records attributed to Weiss, R..

At least 19 recordsLinked to original sources

Generative Neuromorphic Programming of Mammalian Cells

Cells make decisions through densely interconnected regulatory networks that compute graded, analog responses. Synthetic biology has made cells programmable, but the field's dominant abstractions poorly match the analog nature of cells. Designing analog circuits that work in cells is challenging: predicting how components combine remains difficult, and the vast combinatorial space of parts, weights and topologies exceeds what experiments can explore. Here we establish generative programming of mammalian cells through a neuromorphic framework that compiles desired behavior into DNA. Composable regulatory devices built from RNA-targeting endoribonucleases (ERNs) supply signed weights and nonlinear activation, enabling multi-layer circuits with diverse multi-input analog responses. To predict and design such circuits, we develop biomorphic neural networks (BMNs): a compositional architecture that mirrors biological interactions, in which each basic process (transcription, translation, cleavage) is captured by a reusable neural block learned from the behavior of whole circuits that contain it. Recomposed into architectures absent from training, these models predict responses with errors often comparable to variability between experimental repeats. Inverting this process, our software package, the biocompiler, jointly optimizes topology, parts, and weights to realize specified target behaviors. Challenged with three target behaviors, the biocompiler proposed three previously unseen architectures; we built all three, and each reproduced its target behavior in mammalian cells in a single design pass without manual tuning. Generative neuromorphic programming thus offers a systematic route to engineering the analog computation native to living cells.

synthetic biology↗

CRISPR activation screens identify core protein-dependent regulation of heparan sulfate sulfation and ligand specificity

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3-O-sulfated HS motifs, or the N-sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6-O-sulfation and serve as superior substrates for 3-O-sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

Cell Biology↗

A food chain of ribozymes built on an ancestral strand

Molecular life forms in the prebiotic earth were capable of self-reproduction from basic building blocks. However, scarcity of building blocks likely led to the evolution of heterotrophic, predatory species. In this work we reconstruct a simple model of such interactions between RNA molecules, based on RNA strands found in sinkhole mineral pools in the Dead Sea. These RNA strands exhibited significant stability in this divergent chemical environment, and contained sequence motifs reminiscent of two ribozyme species, suggesting they could be ancestral to both. We built a system in which a single ancestral RNA strand serves as a limiting resource forming two different species of ribozymes that compete over it. One of these species is capable of metabolizing the other in order to free more of this resource, driving the assembly of additional copies of itself. This interaction resembles a rudimentary food chain, the dynamics of which is sensitive to environmental factors such as temperature and magnesium concentrations, with competitive advantage shifting between the species. Taken together, these findings highlight a hypothetical feature of prebiotic life, and hint at the possibility that heterotrophic RNA is a phenomenon that might still occur today in some unique geochemical niches, and even in the overlooked background of modern biological systems.

biophysics↗

Reversing transgene silencing via targeted chromatin editing

Mammalian cell engineering offers the opportunity to uncover biological principles and develop next-generation biotechnologies. However, epigenetic silencing of transgenes hinders the control of gene expression in mammalian cells. Here, we use chromatin editing of an integrated reporter in CHO-K1 and human induced pluripotent stem cells to study the molecular interactions driving silencing and its reversal. After transient induction of either DNA methylation or H3K9me3, stable silencing was exclusively observed with both marks. Due to the positive feedback between DNA methylation and H3K9me3 and the relative low stability of H3K9me3, our model predicts that removing DNA methylation is sufficient for transgene reactivation. Accordingly, targeted DNA demethylation reactivated the reporter irrespective of whether silencing was achieved by inducing DNA methylation, H3K9me3, or by the endogenous cellular machinery. These results shed light on molecular mechanisms at play during silencing and provide engineering tools for potent and specific transgene reactivation in mammalian cells.

bioengineering↗

ETV2 mediated differentiation of human pluripotent stem cells results in functional endothelial cells for engineering advanced vascularized microphysiological models

Patient-specific microphysiological models, exemplified by organs-on-a-chip and organoids, have become a valuable tool for broad applications, revolutionizing biomedical research. However, limitations persist, with functional vasculature being a significant challenge. Generating functional human induced pluripotent stem cell (h-iPSC) derived endothelial cells (h-iECs) represent an urgent need. With the discovery of ETV2s determinant role in specifying EC lineages during differentiation, researchers have adopted techniques involving ETV2 overexpression to produce h-iECs more efficiently and consistently. However, the capacity of these cells to form functional vasculatures has not yet been thoroughly investigated. Here, we generated multiple h-iPSC lines with inducible ETV2 expression, and subsequently differentiated them into h-iECs, which were validated functionally and by key endothelial markers and RNA-seq analysis. These cells are capable of self-organizing into stable microvascular networks (MVNs) in a microfluidic chip reproducibly, forming lumenized and functional vessels that mimic the in vivo capillary bed in both morphology and function - a result not achieved using h-iECs differentiated with conventional two-step methods using the same h-iPSC lines. Furthermore, complex microphysiological models featuring perfusable vasculature were also successfully developed using ETV2 activated h-iECs, demonstrated with vascularized tumor and blood-brain barrier (BBB) models. Additionally, by pooling genetically engineered h-iPSCs with inducible ETV2, we effectively employed an orthogonally induced differentiation approach to enhance vascularization of an organoid model. Our methodology opens avenues in precision medicine, leading to personalized microphysiological models with perfusable vasculature for various applications.

bioengineering↗

Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug- regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

bioengineering↗

Magnetized Cellbots to Spatiotemporally Control Differentiation of Human-Induced Pluripotent Stem Cells

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 mature tissue structures as needed. Our work introduces a magnetically controlled microrobot (MR) platform for guiding mammalian cells to desired locations that, combined with synthetic biology, delivers biological signals at precise locations and times, enabling spatiotemporal control of cell-fate decision-making. We use synNotch, a cell-cell contact-based biological signaling that induces relevant gene expression in receivers when the receiver cells contact sender cells through ligand-receptor binding. Magnetically driven MRs are then allowed to be internalized by sender cells, resulting in magnetized sender cellbots. Using a 3-pair orthogonal Helmholtz coil system, we guided magnetized sender cellbots to precise locations in a receiver cell culture, activating desired fluorescent protein expression in target Chinese Hamster Ovary (CHO) receiver cells. Next, we engineered Human-Induced Pluripotent Stem Cells (hiPSC) to function as receivers that can be instructed by senders to differentiate into endothelial cells (ECs) via overexpression of ETV2 (ETS variant transcription factor 2), a master transcriptional regulator of endothelial cell development. Using our magnetic platform, we guided multiple sender cellbots to target locations on a monolayer of hiPSC receivers, resulting in differentiation of receivers into ECs and possible onset of vascular formation. Our approach provides a foundation for the engineering spatial patterns by activating conditional triggers based on MR location and cell state at multiple time points, enabling several applications such as control of organoid architecture.

synthetic biology↗

Divergent regulatory element programs steer sex-specific supporting cell differentiation along mouse gonadal development

Gonadal sex determination relies on tipping a delicate balance involving the activation and repression of several transcription factors and signalling pathways. This is likely mediated by numerous non-coding regulatory elements that shape sex-specific transcriptomic programs. To explore the dynamics of these in detail, we performed paired time-series of transcriptomic and chromatin accessibility assays on pre-granulosa and Sertoli cells throughout their development in the embryo, making use of new and existing mouse reporter lines. Regulatory elements were associated with their putative target genes by linkage analysis, and this was complemented and verified experimentally using promoter capture Hi-C. We identified the transcription factor motifs enriched in these regulatory elements along with their occupancy, pinpointing LHX9/EMX2 as potentially critical regulators of ovarian development. Variations in the DNA sequence of these regulatory elements are likely to be responsible for many of the unexplained cases of individuals with Differences of Sex Development. TeaserMultiomics analysis revealed the regulatory elements and transcription factors responsible for gonadal sex determination.

developmental biology↗

A Genetic Toggle Switch in Plants

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 for devices in plants has lagged. Programmable genetic devices require defining the components quantitative functions. Because plants have long life spans, studies often use transient analysis to define quantitative functions while verification in stably engineered plants is often neglected and largely unknown. This raises a question if unique attributes of plants such as environmental sensitivity, developmental plasticity, or alternation of generations, adversely impacts predictability of plant genetic circuits and devices. Alternatively, it is also possible that genetic elements to produce predictable genetic devices for plants require rigorous characterization with detailed mathematical modeling. Here we use plant genetic elements with quantitatively characterized transfer functions and developed in silico models to guide their assembly into a genetic device: a toggle switch or a mutually inhibitory gene-regulatory device. Our approach allows computational selection of plant genetic components and iterative refinement of the circuit if the desired genetic functions are not initially achieved. We show that our computationally selected genetic circuit functions as predicted in stably engineered plants including through tissue and organ differentiation. Developing abilities to produce predictable and programmable plant genetic devices opens the prospect of predictably engineering plants unique abilities in sustainable human and environmental systems.

synthetic biology↗

The restricted N-glycome of neurons is programmed during differentiation

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 processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies. To understand cell-specific contributions to the unique brain N-glycome and its abundance of Man-5, we performed RNAseq and MALDI-MS TOF protein N-glycomics at several timepoints during differentiation of multiple cell types. To this end, homogeneous cultures of glutamatergic neurons, GABAergic neurons, and brain-specific endothelial cells were generated from monoclonal human inducible pluripotent stem cells (hiPSCs) through cellular reprogramming. Small molecule induction of stably integrated synthetic transcription units driving morphogen expression generated differentiated cells with distinct patterns mirroring intact tissue. Comparing uninduced hiPSCs for each cell type revealed identical transcriptomic and glycomic profiles before differentiation, with low quantities of Man-5. In differentiated glutamatergic and GABAergic neurons, the most abundant N-glycans became Man-5 and its immediate precursor Man-6, despite the presence of transcripts encoding enzymes for their subsequent modification. Differentiation to brain-specific endothelial cells showed an opposite effect, with the N-glycome displaying an abundance of complex N-glycans and terminal modifications of the late secretory pathway. These results confirm that the restricted N-glycome profile of brain is programmed into neuronal differentiation, with regulation independent of the transcriptome and under tight evolutionary constraint.

neuroscience↗

Janus Mazebots and Cellbots Navigating Obstacles in Dense Mammalian Environment

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 microrobots in densely populated environments. In this paper, we fabricate the Mazebot microrobots using silica spheres and ferromagnetic coating, and we use them to create cellbots with genetically modified Chinese Hamster Ovary (CHO) cells. Subsequently, we navigate both the Mazebots and the cellbots through a dense environment populated by CHO cells. The Mazebots navigation is done with a control system that allows the Mazebots to swim on their own, or guide a specific cell from a given origin to a target location while avoiding cell obstacles. The control system operates in open and closed-loop modes, where the first one allows the microrobot to reorient the cell using self-induced fluid vortices, and the second one closely follows a predefined trajectory along the origin and destination. On the other hand, the cellbots navigation is done in closed-loop operation. This enables cell manipulation for potential applications in cell and tissue engineering when in a confined space. Biocompatibility of the Mazebots is confirmed through the exposure of CHO cells to the robots for 24 hours. Experimental results demonstrate the functionality of our algorithm and its potential for biomedical applications, showcasing our system as a powerful and efficient solution for precise cellular manipulation.

bioengineering↗

Analog epigenetic cell memory by graded DNA methylation

Chemical modifications to histones and DNA play a crucial role in the regulation of transcription and in the maintenance of chromatin states that are not permissive to gene expression [1-3]. However, the landscape of gene expression states that these modifications stably maintain remains uncharted. Here, we show that gene expression can be memorized at a wide range of levels thus implementing analog epigenetic memory. Mechanistically, we find that DNA methylation serves a primary role in maintaining memory across cell divisions while histone modifications only follow DNA methylation to regulate gene expression. Employing targeted epigenetic editing and time-course analysis, we analyzed the temporal stability of gene expression and DNA methylation post removal of epigenetic effectors. We found that the grade of DNA methylation in the genes promoter, defined as the mean fraction of methylated CpGs, remains stable over time and inversely correlates with gene expression level. By contrast, Histone 3 lysine 9 trimethylation (H3K9me3) could not persist after removal of its writer in the absence of DNA methylation. These experimental findings, combined with our chromatin modification model, indicate that the absence of positive feedback mechanisms around DNA methylation - unlike those found in histone modifications - enable the temporal stability of the DNA methylation grade, which leads to analog memory. These results expand current knowledge on how epigenetic memory is achieved in natural systems. Moreover, we anticipate that analog memory through graded DNA methylation will enable to program mammalian cells with fine-grained information storage. This capability will significantly enhance the sophistication of engineered cell functionality in applications including tissue engineering, organoids, and cell therapies.

bioengineering↗

Heparan sulfate modified proteins affect cellular processes central to neurodegeneration and modulate presenilin function

Mutations in presenilin-1 (PSEN1) are the most common cause of familial, early-onset Alzheimers disease (AD), typically producing cognitive deficits in the fourth decade. A variant of APOE, APOE3 Christchurch (APOE3ch), was found associated with protection from both cognitive decline and Tau accumulation in a 70-year-old bearing the disease-causing PSEN1-E280A mutation. The amino acid change in ApoE3ch is within the heparan sulfate (HS) binding domain of APOE, and purified APOEch showed dramatically reduced affinity for heparin, a highly sulfated form of HS. The physiological significance of ApoE3ch is supported by studies of a mouse bearing a knock-in of this human variant and its effects on microglia reactivity and A{beta}-induced Tau deposition. The studies reported here examine the function of heparan sulfate-modified proteoglycans (HSPGs) in cellular and molecular pathways affecting AD-related cell pathology in human cell lines and mouse astrocytes. The mechanisms of HSPG influences on presenilin-dependent cell loss and pathology were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss of function of sulfateless (sfl), a homolog of NDST1, a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in cultured human cell lines, Drosophila, and mouse astrocytes. RNAi of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. RNA-sequence analysis of human cells deficient in HS synthesis demonstrated effects on the transcriptome governing lipid metabolism, autophagy, and mitochondrial biogenesis and showed significant enrichment in AD susceptibility genes identified by GWAS. Neuron-directed knockdown of Psn in Drosophila produced cell loss in the brain and behavioral phenotypes, both suppressed by simultaneous reductions in sfl mRNA levels. Abnormalities in mitochondria, liposome morphology, and autophagosome-derived structures in animals with Psn knockdown were also rescued by simultaneous reduction of sfl. sfl knockdown reversed Psn-dependent transcript changes in genes affecting lipid transport, metabolism, and monocarboxylate carriers. These findings support the direct involvement of HSPGs in AD pathogenesis.

neuroscience↗

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts

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 pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications. One-sentence summaryGenetic controllers and live-cell imaging offer a versatile strategy for probing the role of transcription factor dynamics in cell fate transitions.

synthetic biology↗

A Remarkable Adaptive Paradigm Of Heart Performance And Protection Emerges In Response To The Constitutive Challenge Of Marked Cardiac-Specific Overexpression Of Adenylyl Cyclase Type 8

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload ([~]30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide array of biological processes and molecular functions in numerous cellular compartments differed in TGAC8 vs WT; and over 250 canonical signaling pathways characteristic of adaptive survival circuitry of cancers, including PI3K and growth factor signaling, cytokine and T cell receptor signaling, immune responses, ROS scavenging, proliferation, protection from apoptosis, and nutrient sensing, were activated in TGAC8; and compared to WT there was a shift from fatty acid oxidation to increased aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, the adaptive paradigm, that becomes activated in the LV of TGAC8 in response to severe chronic, intense AC/PKA/Ca2+ signaling embodies many hallmarks of cancer.

physiology↗

Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks

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 models, however, depends on the accuracy of their predictions, the precision of the measurements used to parameterize the models, and the tolerance of biological devices for imperfections in modeling and measurement. To better understand this relationship, we have derived an Engineering Error Inequality that provides a quantitative mathematical bound on the relationship between predictability of results, model accuracy, measurement precision, and device characteristics. We apply this relation to estimate measurement precision requirements for engineering genetic regulatory networks given current model and device characteristics, recommending a target standard deviation of 1.5-fold. We then compare these requirements with the results of an interlaboratory study to validate that these requirements can be met via flow cytometry with matched instrument channels and an independent calibrant. Based on these results, we recommend a set of best practices for quality control of flow cytometry data and discuss how these might be extended to other measurement modalities and applied to support further development of genetic regulatory network engineering.

synthetic biology↗

Allergy-induced systemic inflammation impairs tendon quality

Treatment of tendinopathies still present a major challenge, since the aetiology of the disease remains poorly understood. To determine whether the systemic inflammation accompanying predisposing factors including rheumatoid arthritis, diabetes or smoking contributes to the onset of tendinopathy, we studied the effect of a systemic inflammation induced by an allergic episode on tendon properties. To this end, we elicited an allergic response in mice by exposing them to a plant allergen and subsequently analysed both their flexor and Achilles tendons. Biomechanical testing and histological analysis revealed that tendons from allergic mice not only showed a significant reduction of both elastic modulus and tensile stress, but also alterations of the tendon matrix. Moreover, 3D tendon-like constructs treated with sera from allergic mice displayed a matrix-remodelling expression profile and the expression of macrophage-associated markers and matrix metalloproteinase 2 (MMP2) was increased in allergic Achilles tendons. Analysing data from an epidemiologic study comprising data from more than 10.000 persons, we found that persons suffering from an allergic condition appeared to have an increased propensity to develop a tendinopathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/450910v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1263836org.highwire.dtl.DTLVardef@349621org.highwire.dtl.DTLVardef@6c9142org.highwire.dtl.DTLVardef@1b49bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Laser facilitated epicutaneous peptide immunization using dry patch technology

The skin has been intensely investigated as a target tissue for immunization because it is populated by multiple types of antigen presenting cells. Directly addressing dendritic cells or Langerhans cells in vivo represents an attractive strategy for inducing T cell responses in cancer immunotherapy. We and others have studied fractional laser ablation as a novel method combining efficient delivery of macromolecules to the skin with an inherent adjuvant effect of laser illumination. In this proof of concept study, we demonstrate the feasibility of peptide delivery to the skin using the P.L.E.A.S.E. professional Erb:YAG fractional infrared laser together with EPIMMUN patches. In an ovalbumin mouse model we demonstrate that a dry patch formulation of SIINFEKL peptide in combination with CpG-ODN1826, but not imiquimod or polyI:C, induces potent cytotoxic T cell responses, which can be further boosted by co-delivery of the pan-helper T cell epitope PADRE.

immunology↗