Biology subjects

Kamat, N. P.

Publications and source records attributed to Kamat, N. P..

10 recordsLinked to original sources

bioRxiv2026

CELLISA - a cell-cell binding assay for evaluation of nanovesicle targeting proteins

Enhancing targeted delivery of biomedicines improves efficacy and can reduce off-target effects by lowering the effective dose, but achieving targeting is challenging. Extracellular vesicles (EVs) are promising biological nanovesicles which can be targeted by displaying binding proteins and are being developed as therapeutics. Currently, discovering EV targeting constructs is limited by low throughput and resource-intensive EV production and isolation. To accelerate discovery, we developed a screening pipeline to identify EV targeting constructs without requiring EV production. This approach is premised on the hypothesis that cell-cell interactions may predict some cell-EV interactions. Our cell binding assay (CELLISA) quantifies binding of a cell surface-displayed targeting protein to its cognate receptor on a target cell, employing a microscopy-based analysis pipeline. After validating the premise using existing T cell-targeting reagents, we develop CELLISA for either adherent or suspension EV producer cells. Finally, we use CELLISA to evaluate new binders and validate that hits mediate targeting and/or delivery of genetic cargo to natural killer cells and T cells. CELLISA increased throughput > 6-fold and decreased time by 40% compared to standard EV screens, and it identified a T-cell binder conferring efficient gene delivery. CELLISA is easily adaptable to other laboratories and can accelerate EV research.

bioengineering↗

bioRxiv2025

DNA-Directed Assembly of Multivalent Lipid Nanoparticles for Targeted T Cell Gene Delivery

Lipid nanoparticles (LNPs) are a powerful emerging tool for in vivo T cell engineering with applications ranging from B cell lymphomas to other cancers and autoimmune diseases. Key challenges in designing these therapeutics include achieving both precise cell targeting and efficient mRNA translation. While single-targeted LNPs have been extensively studied, bispecific LNPs have only been briefly explored. Engagement of multiple T cell receptors offers the opportunity for enhanced mRNA delivery, expression, and T cell targeting. Here, a DNA-tethering method enables rapid modification of lipid nanoparticles with commercial antibodies. Using this strategy, we evaluated a variety of bispecific LNPs for targeted mRNA delivery to T cells both in vitro and in vivo. We identify bispecific formulations that improve targeting and subsequent transfection of T cells in vitro and in vivo relative to monotargeted LNPs. Additionally, we find that targeting molecules can alter LNP biodistribution to the spleen and liver. This fast and efficient approach to assembling antibody-targeted LNPs should enable high-throughput screening of diverse antibody combinations for improved specificity and efficiency of in vivo gene delivery. Significance StatementA major challenge in in vivo gene delivery is achieving both precise targeting and efficient mRNA translation. Multitargeted LNPs offer a potential solution to this challenge; however, their rapid assembly remains difficult, necessitating the development of new methods to construct and evaluate targeted LNPs. Here, we use a DNA-tethering method to enable rapid antibody modification of LNPs and evaluate bispecific formulations for targeted T cell mRNA delivery in vitro and in vivo. We find bispecific LNPs improve T cell targeting and expression compared to single-targeted particles. To the best of our knowledge, this study represents the first systematic screening and comparison of bispecific LNPs. Our method provides a modular approach for identifying effective antibody combinations to enhance in vivo gene delivery that can be customized for different undruggable diseases.

immunology↗

bioRxiv2025

Distinguishing Protein and Gene Delivery Enables Characterization and Bioengineering of Extracellular Vesicle-Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) gene therapies have achieved some clinical success, with multiple products reaching regulatory approval. Encapsulation of AAV vectors within engineered extracellular vesicles (EVs) is an emerging strategy which could help overcome challenges including pre-existing anti-capsid immunity and the need for controlling targeting and tropism. To guide the development of EV-AAV technologies, we developed an assay for quantifying and controlling for the contribution of pseudotransduction to evaluations of EV-AAV-mediated transduction. We developed an AAV vector that switches its transgene output from one reporter to another when acted upon by Cre recombinase expressed in a recipient cell. Using this platform, we investigated EV-AAV transduction as a function of various engineered EV surface modifications. For actively endocytic cells (HEK293FTs), modifications that enhance EV uptake and membrane fusion influence pseudotransduction but not true transduction. Conversely, in less endocytic Jurkat T cells, modifications enhancing EV uptake enhanced both pseudotransduction and true transduction. These conclusions held across two AAV serotypes. Our results provide new insight into prior reports and suggest that effects of enhancing uptake and membrane fusion of EV-AAV vectors are recipient cell type-specific. The methods developed here unambiguously dissect EV-AAV transduction mechanisms and can guide future bioengineering of EV-AAV vectors.

bioengineering↗

bioRxiv2024

Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design

Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.

synthetic biology↗

bioRxiv2023

Remodeling of lipid-foam prototissues by network-wide tension fluctuations induced by active particles

Recent advances in the field of bottom-up synthetic biology have led to the development of synthetic cells that mimic some features of real cells, such as division, protein synthesis, or DNA replication. Larger assemblies of synthetic cells may be used to form prototissues. However, existing prototissues are limited by their relatively small lateral dimensions or their lack of remodeling ability. In this study, we introduce a lipid-based tissue mimetic that can be easily prepared and functionalized, consisting of a millimeter-sized "lipid-foam" with individual micrometer-sized compartments bound by lipid bilayers. We characterize the structural and mechanical properties of the lipid-foam tissue mimetic. We demonstrate self-healing capabilities enabled by the fluidity of the lipid bilayers. Upon inclusion of bacteria in the tissue compartments, we observe that the tissue mimetic exhibits network-wide tension fluctuations driven by membrane tension generation by the swimming bacteria. Active tension fluctuations facilitate the fluidization and reorganization of the lipid-foam tissue mimetic, providing a versatile platform for understanding and mimicking biological tissues.

synthetic biology↗

bioRxiv2023

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

synthetic biology↗

bioRxiv2022

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The extent to which membrane biophysical properties, such as hydrophobic thickness, can drive membrane protein organization remains unknown. Inspired by this question, we used de novo protein design, molecular dynamic simulations, and cell-free systems to elucidate how membrane-protein hydrophobic mismatch affects protein integration and organization in synthetic lipid membranes. We found that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We used this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to self-organizing membrane-based materials with new functions.

synthetic biology↗

bioRxiv2022

Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. By encapsulating these systems in synthetic membranes, we can reintroduce features of the cell membrane, including molecular containment and selective permeability, which together could significantly enhance sensing capabilities. Here, we demonstrate robust and tunable performance of an encapsulated fluoride riboswitch inside of lipid vesicles. The riboswitch remains active upon encapsulation in lipid vesicles and responds to fluoride added to the surrounding solution. We find the sensitivity of the encapsulated sensor can be tuned by varying membrane composition. We then show that encapsulation protects the sensor from degradation by the sample and use two types of genetically encoded outputs to detect fluoride in real-world samples. This work establishes the feasibility of vesicle-encapsulated cell-free systems to detect environmentally relevant small molecules.

synthetic biology↗

bioRxiv2020

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

synthetic biology↗

bioRxiv2019

Barcoding biological reactions with DNA-functionalized vesicles

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

synthetic biology↗