Synthetic biology in the clinic: engineering vaccines, diagnostics, and therapeutics.

Synthetic biology is a design-driven discipline centered on engineering novel biological functions through the discovery, characterization, and repurposing of molecular parts. Several synthetic biological solutions to critical biomedical problems are on the verge of widespread adoption and demonstrate the burgeoning maturation of the field. Here, we highlight applications of synthetic biology in vaccine development, molecular diagnostics, and cell-based therapeutics, emphasizing technologies approved for clinical use or in active clinical trials. We conclude by drawing attention to recent innovations in synthetic biology that are likely to have a significant impact on future applications in biomedicine.

Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses.

T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical "upgrades," such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system.

Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin1,2, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the ß-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Topical dorzolamide for macular holes: A randomised, double-blind, placebo-controlled trial.

BACKGROUND: To assess topical dorzolamide as medical therapy for idiopathic full-thickness macular holes (FTMHs). METHODS: Randomised, double-blinded, placebo-controlled, single-centre clinical trial involving 32 patients with idiopathic small FTMHs (<400 µm $$ \upmu \mathrm{m} $$ ). Participants in both arms used topical dorzolamide 2% or saline thrice daily for 8 weeks with monthly OCT. Those with persisting FTMH underwent vitrectomy with ILM peel and gas tamponade. The primary outcome was the rate of FTMH closure at the end of treatment. RESULTS: Between 6 March 2020 and 16 June 2023, 32 eligible patients were enrolled: 16 participants in each arm. All participants in both groups were included in the final analysis. At the final visit, 3 of 16 (18.8%) patients in both the topical dorzolamide and placebo group demonstrated closure. There was no statistically significant difference in the proportion of FTMH closure between the control and treatment group (p = 1.00), nor statistically significant difference in the mean change in best corrected visual acuity (BCVA; p = 0.909). There was no difference in the change in FTMH diameter between groups (p = 0.225). No serious adverse events were reported in either group. CONCLUSION: Topical dorzolamide was safe but not superior to placebo in the functional and anatomical outcomes of FTMH.

Short-chain fatty acid concentrations in the incidence and risk-stratification of colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The beneficial role of gut microbiota and bacterial metabolites, including short-chain fatty acids (SCFAs), is well recognized, although the available literature around their role in colorectal cancer (CRC) has been inconsistent. METHODS: We performed a systematic review and meta-analysis to examine the associations of fecal SCFA concentrations to the incidence and risk of CRC. Data extraction through Medline, Embase, and Web of Science was carried out from database conception to June 29, 2022. Predefined inclusion/exclusion criteria led to the selection of 17 case-control and six cross-sectional studies for quality assessment and analyses. Studies were categorized for CRC risk or incidence, and RevMan 5.4 was used to perform the meta-analyses. Standardized mean differences (SMD) with 95% confidence intervals (CI) were calculated using a random-effects model. Studies lacking quantitation were included in qualitative analyses. RESULTS: Combined analysis of acetic, propionic, and butyric acid revealed significantly lower concentrations of these SCFAs in individuals with a high-risk of CRC (SMD = 2.02, 95% CI 0.31 to 3.74, P = 0.02). Additionally, CRC incidence was higher in individuals with lower levels of SCFAs (SMD = 0.45, 95% CI 0.19 to 0.72, P = 0.0009), compared to healthy individuals. Qualitative analyses identified 70.4% of studies reporting significantly lower concentrations of fecal acetic, propionic, butyric acid, or total SCFAs in those at higher risk of CRC, while 66.7% reported significantly lower concentrations of fecal acetic and butyric acid in CRC patients compared to healthy controls. CONCLUSIONS: Overall, lower fecal concentrations of the three major SCFAs are associated with higher risk of CRC and incidence of CRC.

Postpartum circulating microRNA enhances prediction of future type 2 diabetes in women with previous gestational diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes mellitus is a major cause of morbidity and death worldwide. Women with gestational diabetes mellitus (GDM) have greater than a sevenfold higher risk of developing type 2 diabetes in later life. Accurate methods for postpartum type 2 diabetes risk stratification are lacking. Circulating microRNAs (miRNAs) are well recognised as biomarkers/mediators of metabolic disease. We aimed to determine whether postpartum circulating miRNAs can predict the development of type 2 diabetes in women with previous GDM. METHODS: In an observational study, plasma samples were collected at 12 weeks postpartum from 103 women following GDM pregnancy. Utilising a discovery approach, we measured 754 miRNAs in plasma from type 2 diabetes non-progressors (n = 11) and type 2 diabetes progressors (n = 10) using TaqMan-based real-time PCR on an OpenArray platform. Machine learning algorithms involving penalised logistic regression followed by bootstrapping were implemented. RESULTS: Fifteen miRNAs were selected based on their importance in discriminating type 2 diabetes progressors from non-progressors in our discovery cohort. The levels of miRNA miR-369-3p remained significantly different (p < 0.05) between progressors and non-progressors in the validation sample set (n = 82; 71 non-progressors, 11 progressors) after adjusting for age and correcting for multiple comparisons. In a clinical model of prediction of type 2 diabetes that included six traditional risk factors (age, BMI, pregnancy fasting glucose, postpartum fasting glucose, cholesterol and triacylglycerols), the addition of the circulating miR-369-3p measured at 12 weeks postpartum improved the prediction of future type 2 diabetes from traditional AUC 0.83 (95% CI 0.68, 0.97) to an AUC 0.92 (95% CI 0.84, 1.00). CONCLUSIONS: This is the first demonstration of miRNA-based type 2 diabetes prediction in women with previous GDM. Improved prediction will facilitate early lifestyle/drug intervention for type 2 diabetes prevention.

Strength of T cell signaling regulates HIV-1 replication and establishment of latency.

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir.

The dendritic cell receptor Clec9A binds damaged cells via exposed actin filaments.

The immune system must distinguish viable cells from cells damaged by physical and infective processes. The damaged cell-recognition molecule Clec9A is expressed on the surface of the mouse and human dendritic cell subsets specialized for the uptake and processing of material from dead cells. Clec9A recognizes a conserved component within nucleated and nonnucleated cells, exposed when cell membranes are damaged. We have identified this Clec9A ligand as a filamentous form of actin in association with particular actin-binding domains of cytoskeletal proteins. We have determined the crystal structure of the human CLEC9A C-type lectin domain and propose a functional dimeric structure with conserved tryptophans in the ligand recognition site. Mutation of these residues ablated CLEC9A binding to damaged cells and to the isolated ligand complexes. We propose that Clec9A provides targeted recruitment of the adaptive immune system during infection and can also be utilized to enhance immune responses generated by vaccines.

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits.

Boolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.

Neutralising antibodies block the function of Rh5/Ripr/CyRPA complex during invasion of Plasmodium falciparum into human erythrocytes.

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine.

Chronic Rejection of Cardiac Allografts Is Associated With Increased Lymphatic Flow and Cellular Trafficking.

BACKGROUND: Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. METHODS: Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. RESULTS: Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4+, CD8+ T cells, and CD68+ macrophages. CONCLUSIONS: Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.

Synthetic Biology: Immunotherapy by Design.

Cellular immunotherapy holds great promise for the treatment of human disease. Clinical evidence suggests that T cell immunotherapies have the potential to combat cancers that evade traditional immunotherapy. Despite promising results, adverse effects leading to fatalities have left scientists seeking tighter control over these therapies, which is reflected in the growing body of synthetic biology literature focused on developing tightly controlled, context-independent parts. In addition, researchers are adapting these tools for other uses, such as for the treatment of autoimmune disease, HIV infection, and fungal interactions. We review this body of work and devote special attention to approaches that may lend themselves to the development of an "ideal" therapy: one that is safe, efficient, and easy to manufacture. We conclude with a look toward the future of immunotherapy: how synthetic biology can shift the paradigm from the treatment of disease to a focus on wellness and human health as a whole.

Expression of miR-206 in human islets and its role in glucokinase regulation.

Inappropriate insulin secretion from ß-cells is considered as an early sign of impaired glucose tolerance and type 2 diabetes (T2D). Glucokinase (GCK) is an important enzyme that regulates glucose metabolism and ensures that the normal circulating glucose concentrations are maintained. GCK expression is induced by glucose and regulated via transcription factors and regulatory proteins. Recently, microRNA-206 (miR-206) was reported to regulate GCK and alter glucose tolerance in normal and high-fat diet-fed mice. Although the study findings have implications for human diabetes, studies in human islets are lacking. Here, we analyze human islets from individuals without or with T2D, using TaqMan-based real-time qPCR at the tissue (isolated islet) level as well as at single cell resolution, to assess the relationship between miR-206 and GCK expression in normal and T2D human islets. Our data suggest that, unlike mouse islets, human islets do not exhibit any correlation between miR-206 and GCK transcripts. These data implicate the need for further studies aimed toward exploring its potential role(s) in human islets.

Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells.

Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection. Although these effectors contribute to pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behaviour. Here we exploit two effector proteins, the Shigella flexneri OspF protein and Yersinia pestis YopH protein, to rewire kinase-mediated responses systematically both in yeast and mammalian immune cells. Bacterial effector proteins can be directed to inhibit specific mitogen-activated protein kinase pathways selectively in yeast by artificially targeting them to pathway-specific complexes. Moreover, we show that unique properties of the effectors generate new pathway behaviours: OspF, which irreversibly inactivates mitogen-activated protein kinases, was used to construct a synthetic feedback circuit that shows novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to tune the T-cell response amplitude precisely, or as an inducible pause switch that can temporarily disable T-cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.

Synthesis, Characterization, and Application of Core-Shell Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm) Nanoparticle as Trimodal (MRI, PET/SPECT, and Optical) Imaging Agents.

Multimodal nanoparticulate materials are described, offering magnetic, radionuclide, and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography/single-photon emission commuted tomography (PET/SPECT), and optical imaging. They comprise Fe3O4@NaYF4 core/shell nanoparticles (NPs) with different cation dopants in the shell or core, including Co0.16Fe2.84O4@NaYF4(Yb, Er) and Fe3O4@NaYF4(Yb, Tm). These NPs are stabilized by bisphosphonate polyethylene glycol conjugates (BP-PEG), and then show a high transverse relaxivity (r2) up to 326 mM(-1) s(-1) at 3T, a high affinity to [(18)F]-fluoride or radiometal-bisphosphonate conjugates (e.g., (64)Cu and (99m)Tc), and fluorescent emissions from 500 to 800 nm under excitation at 980 nm. The biodistribution of intravenously administered particles determined by PET/MR imaging suggests that negatively charged Co0.16Fe2.84O4@NaYF4(Yb, Er)-BP-PEG (10K) NPs cleared from the blood pool more slowly than positively charged NPs Fe3O4@NaYF4(Yb, Tm)-BP-PEG (2K). Preliminary results in sentinel lymph node imaging in mice indicate the advantages of multimodal imaging.

A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface.

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.