Biology subjects

Jerala, R.

Publications and source records attributed to Jerala, R..

11 recordsLinked to original sources

bioRxiv2026

Pharmacologically regulated bioorthogonal stabilization domain for regulation of CAR T cells

Regulation of therapeutic cell response is important for safe and effective therapy, particularly for immunotherapy. Ideally, the regulators should be based on human proteins using compounds that have already been approved for human use. Regulation of protein degradation by small molecules enables fast cellular response and a small genetic footprint of genetic constructs. Here, we present a clinically compatible strategy for reversible pharmacological control of chimeric antigen receptor (CAR) T cell function using human estrogen receptor (ER)-based degron domains and FDA-approved small molecules. By fusing tamoxifen-responsive ER ligand-binding domains to CARs, we generate ligand-inducible ON-switch CARs whose stability, signaling, and effector functions are precisely controlled by 4-hydroxytamoxifen. Furthermore, we demonstrate that ER-tagged CARs can be selectively degraded using the FDA-approved ER-targeting PROTAC ARV-471, establishing a complementary OFF-switch mechanism that suppresses CAR expression and effector function. Notably, these results expand the bioorthogonal ON-OFF switch platform for post-translational control of therapeutic proteins, offering new opportunities to improve the safety and precision of cellular immunotherapies.

synthetic biology↗

bioRxiv2025

Hydroxychloroquine decouples reactogenicity from efficacy in mRNA and viral-vector gene delivery

Programmable nucleic-acid therapeutics, including lipid nanoparticle (LNP)-formulated mRNA vaccines, genome editors delivered as mRNA, and viral vectors, are transforming precision medicine but remain constrained by innate reactogenicity despite the introduction of chemical modifications into mRNA. Here, we show that mRNA/LNPs and viral vectors elicit a transient inflammatory burst that peaks [~]6 h after dosing in vivo. Co-administration or prophylaxis with hydroxychloroquine (HCQ), a clinically established 4-aminoquinoline, potently attenuated cytokine and chemokine induction across modalities, including strong responses to unmodified mRNA, without compromising therapeutic efficacy. Humoral and cellular immunity to SARS-CoV-2 Spike mRNA vaccines (BNT162b2 and non-modified mRNA/LNP) were preserved, as was live-virus neutralization. Transcriptomic profiling indicated selective dampening of TLR/cGAS-STING pathways with retention of type-I interferon elements compatible with effective vaccination. HCQ further mitigated AAV9-associated blood-brain barrier (BBB) disruption after stereotactic delivery to the mouse brain and reduced mRNA/LNP-associated hepatotoxicity and thrombocytopenia while maintaining therapeutic transgene expression and CRISPR base-editing in vivo. These findings identify HCQ as an anti-reactogenic adjunct that widens the safety window of nucleic-acid therapeutics without sacrificing performance.

immunology↗

bioRxiv2025

Disrupting Splicing Regulation to Rescue β-Catenin: A Novel Approach for Treating CTNNB1-Haploinsufficiency Disorder

Loss-of-function mutations in the CTNNB1 gene cause {beta}-catenin deficiency, resulting in CTNNB1 syndrome--a rare neurodevelopmental disorder characterized by motor and cognitive impairments. Given the wide variety of mutations across CTNNB1 and its dosage sensitivity, a mutation-independent therapeutic approach that preserves endogenous gene regulation is critically needed. This study introduces spliceosome-mediated RNA trans-splicing as a novel approach to restore {beta}-catenin production. Precursor mRNA trans-splicing molecules (PTMs) targeting CTNNB1 introns 2, 5, and 6 were designed and evaluated using a split fluorescent YFP reporter system. Rationally designed short antisense RNAs, which mask splicing regulatory elements, significantly enhanced PTM-mediated trans-splicing at both RNA and protein levels. Additionally, introducing a self-cleaving ribozyme at the PTMs 5 end further improved trans-splicing efficiency, likely due to increased nuclear retention. CMV promoter-driven PTM expression yielded the highest efficiency. Importantly, successful trans-splicing of the endogenous CTNNB1 transcript confirmed the physiological relevance of this strategy. This study is the first to apply and optimize SMaRT for CTNNB1 correction, providing a promising, mutation-agnostic approach for treating CTNNB1 syndrome.

molecular biology↗

bioRxiv2025

Single-chain permuted proteins for dimerization-based control of protein activity and cellular processes

Strategies for detecting and controlling protein interactions play a critical role in gaining insight into molecular mechanisms of biological processes and for the control of cellular processes. Conditional protein reconstitution allows control of the selected protein function based on the proximity, defined by the genetically fused domain pairs, which may be regulated by chemical or biological signals. This typically requires two protein components in a stoichiometric ratio, which increases the complexity and genetic footprint with split segments often being unstable and prone to aggregation. To overcome this limitation, we developed an approach based on a permuted protein reconstitution by conditional dimerization (PROPER). According to this strategy, the N- and C-terminal domains of selected proteins are swapped and a loop is replaced by a short linker that prevents the functionality of a monomeric protein, which reconstitutes only upon di- or oligomerization, controlled by a genetically fused domain that dimerizes by a chemical signal or senses a dimeric target. This design principle was demonstrated on three proteins with diverse functions: a protease, luciferase, and a cytokine. We demonstrate chemically and biologically inducible systems that enable controllable induction of cell death, virus detection, and immune cell stimulation. The PROPER platform expands the chemical biology toolbox, with the benefits of split proteins accomplished by a single component.

synthetic biology↗

bioRxiv2024

A general strategy to engineer high-performance mammalian Whole-Cell Biosensors

Transcription-based whole-cell biosensors (WCBs) are cells engineered with an analyte-responsive promoter driving the transcription of a reporter gene. WCBs can sense and report on bioactive molecules (analytes) relevant to human health. Designing an analyte-sensitive promoter requires a cumbersome trial-and-error approach and usually results in biosensors with poor performance. Here, we integrated Synthetic Biology with Control Engineering to design, computationally model, and experimentally implement high-performance biosensors in mammalian cells. Our approach, unlike traditional methods, does not rely on optimizing individual components such as promoters and transcription factors. Instead, it uses biomolecular circuits to enhance the biosensors performance despite inherent component flaws. We experimentally implemented eight different biosensors by employing CRISPR-Cas systems, then quantitatively compared their performance and identified one configuration, which we named CASense, that overcomes the limitations of current biosensors. Our approach is generalisable and can be adapted to sense any analyte of interest for which there is an analyte-sensitive promoter, making it a versatile tool for multiple applications. As a proof of concept, we engineered a high-performance biosensor of intracellular copper due to the critical role that copper plays in human health and disease, and to the lack of techniques able to measure intracellular copper levels in living cells. The significance of our work lies in its potential to impact the monitoring of bioactive molecules and chemicals both in vitro and in vivo, which is crucial in areas such as toxicology, drug discovery, disease diagnosis and therapy.

synthetic biology↗

bioRxiv2023

Engineering a Scalable and Orthogonal Platform for Synthetic Communication in Mammalian Cells

The rational design and implementation of synthetic, orthogonal mammalian communication systems has the potential to unravel fundamental design principles of mammalian cell communication circuits and offer a framework for engineering of designer cell consortia with potential applications in cell therapeutics and artificial tissue engineering. We lay here the foundations for the engineering of an orthogonal, and scalable mammalian synthetic intercellular communication platform that exploits the programmability of synthetic receptors and selective affinity and tunability of diffusing coiled-coil (CC) peptide heterodimers. Leveraging the ability of CCs to exclusively bind to a selected cognate receptor, we demonstrate orthogonal receptor activation, as well as Boolean logic computations. Next, we reveal synthetic intercellular communication based on synthetic receptors and secreted multidomain CC ligands and demonstrate a minimal, three-cell population system that can perform distributed AND gate logic. Our work provides a modular and scalable framework for the engineering of complex cell consortia, with the potential to expand the aptitude of cell therapeutics and diagnostics.

synthetic biology↗

bioRxiv2022

Designed allosteric protein logic

Regulation of the activity of proteins enables control of complex cellular processes. Allosteric regulation has been introduced individually into few natural proteins. Here, we present a generally applicable regulation of diverse proteins called INSRTR (inserted peptide structure regulator), based on inserting a short unstructured peptide into a solvent-accessible loop that retains protein function. Function of the target protein can be inactivated by the addition of a peptide that forms a rigid coiled-coil dimer. This platform enables the construction of ON/OFF protein switches, their regulation by small molecules, and Boolean logic functions with a rapid response in mammalian cells. INSRTR can be used to regulate a wide range of proteins, as demonstrated on ten members of protein families with diverse biological activities including enzymes, signaling mediators, DNA binders/transcriptional regulators, fluorescent protein, and antibodies regulating chimeric antigen receptor. INSRTR platform presents an extraordinary potential for regulating biological systems and applications. One sentence summaryAuthors have designed a widely applicable system to activate or inactivate function of diverse proteins or form Boolean logic gates based on formation of a coiled-coil dimer within protein loops and demonstrated its implementation on a range of 10 diverse proteins.

synthetic biology↗

bioRxiv2021

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion based on transcriptional control is rather slow, as proteins requires transcription, translation, followed by the transport from the endoplasmic reticulum (ER) through the conventional protein secretion (CPS) pathway towards the plasma membrane. An alternative faster bypass would be valuable. Here we present two genetically encoded orthogonal secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by cytosolic protease) or inside the ER lumen (lumER, released by ER luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables precise combination of signals into logic functions and was demonstrated on a chemically regulated insulin secretion. Regulation of ER escape represents a platform for the design of fast responsive and tightly-controlled modular and scalable protein secretion system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/464966v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@12beb61org.highwire.dtl.DTLVardef@6a664corg.highwire.dtl.DTLVardef@1621e5borg.highwire.dtl.DTLVardef@b728ac_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO membER and lumER system. By equipping a protein of interest (POI) with an N-terminal signaling sequence, which initiates the transport of proteins into the endoplasmic reticulum (ER), and a C-terminal KDEL ER retention sequence for luminal proteins or a KKXX sequence for transmembrane proteins, we can retain those proteins inside the ER and cis-Golgi apparatus (GA) through retrograde transport. Insertion of a protease cleavage site adjacent to the retention signal allows for the regulated fast secretion through proteolytic cleavage. The membrane bound, ER membrane (membER) and ER-luminal (lumER) systems allow for the controlled secretion of pre-synthesized protein, stored inside the ER. This platform enables release of target proteins several hours faster than systems relying transcription and translation. C_FIG

synthetic biology↗

bioRxiv2020

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

synthetic biology↗

bioRxiv2020

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

Myriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

molecular biology↗

bioRxiv2020

Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and {beta}-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue {beta}-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

immunology↗