Biology subjects

Szablowski, J. O.

Publications and source records attributed to Szablowski, J. O..

15 recordsLinked to original sources

bioRxiv2026

Cell-Based Sensor for Extracellular DNA

Detection of molecules with cell-based sensors allows for conversion of binding events into gene expression outputs. Here, we present a cell-based sensor that can detect extracellular double-stranded DNA. This sensor is based on an engineered receptor which we call Luminescent Ultrasensitive Nucleic Acid Reporter, or LUNAR. LUNAR is based on a recently developed Programmable Antigen-gated G-protein-coupled Engineered Receptor (PAGER). PAGERs are a genetic fusion of an auto-inhibitory peptide, a protein-binding domain, and a modified kappa opioid receptor. PAGERs are gated by two binding events. First, a protein ligand displaces an intramolecular inhibitor, Arodyn, then a second ligand activates the receptor. By replacing the protein-binding domain with a DNA binding zinc finger protein (ZFP) we could detect extracellular DNA in a dose-dependent fashion. Here, we show that first-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells. Future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.

synthetic biology↗

bioRxiv2026

Monitoring Gene Expression in Retina with Synthetic Serum Markers

Gene expression underlies retinal development, function, and pathogenesis. However, monitoring retinal gene expression in vivo is challenging. In this study, we apply synthetic serum markers, called Released Markers of Activity, or RMAs, to quantify gene expression in an intact retina through a simple blood test. We show that RMAs can quantify transduction of multiple retinal cell-types and monitor cell implantation or cell loss. We found RMAs are sensitive enough to measure transduction of rare cell populations, such as retinal ganglion cells and could detect transplantation of as few as 100 stem cells. Expression of RMAs showed no evidence of cell loss or immune activation in the retina. Overall, these findings demonstrate that RMAs are highly sensitive, tissue non-destructive gene expression reporters that can continuously track gene delivery and cell survival in intact retina through a simple blood test.

bioengineering↗

bioRxiv2026

Ultrasound-enhanced retinal delivery of engineered viral vectors

The goal of this study was to examine Focused Ultrasound Blood-Retina Barrier Opening (FUS-BRBO) as a method to deliver gene therapy to the eye. We compared AAV.FUS.3, an engineered variant optimized for FUS-enhanced delivery to the central nervous system, and AAV9 for their utility as retinal gene vectors. To achieve this, mouse eyes were insonated by 4.667 MHz FUS in a custom-designed nosecone and vectors administered intravenously. We found 2.1-fold improvement of transduction of AAV.FUS.3 over AAV9 in the insonated area and 19.5-fold improvement over the uninsonated retina. Within that area, the ganglion cell layer showed the highest levels of transduction at 22% for AAV.FUS.3. We observed no gross tissue damage, change in retinal layer thickness, or increase in inflammation markers within the retina. FUS-BRBO has the potential to non-surgically deliver AAV-based vectors to spatially-defined regions of the retina. AAV.FUS.3 showed significantly improved transduction over AAV9 at the same dose. Further research to improve efficiency of delivery to the outer retina could expand the utility of FUS-BRBO.

bioengineering↗

bioRxiv2025

Modeling synthetic serum marker kinetics for monitoring deep-tissue gene expression.

Serum markers could theoretically enable monitoring of gene expression dynamics with a simple blood draw. However, such markers are typically used to measure long-term changes, such as the progression of disease over multiple days or months. In this theoretical study, we determine the maximum theoretical temporal resolution and precision - the ability to distinguish rapid changes in gene expression and to obtain the true frequency of such changes, respectively. As a model of such processes, we used Released Markers of Activity (RMAs) - a class of synthetic serum markers that are expressed in neurons in the brain but transported into the blood. RMAs are orthogonal to physiological processes and have tunable levels, production rate, and onset time, providing well-defined data for serum marker production, tissue transport, and detection. We explore several scenarios where RMAs were used, including monitoring tissue transduction, changes in endogenous gene expression, and drug-induced marker expression. We demonstrate that the temporal resolution of monitoring primarily depends on the extrinsic noise level of the RMA signal and protein serum half-life. Additionally, we find that decreasing the serum half-life results in improved temporal precision at the cost of signal intensity. To enable broad use of this model, we developed a library and interface for running simulations to approximate marker trajectories to inform experimental design decisions and optimize marker detection. Author SummaryGene expression is a foundational driver of biological processes. We use synthetic serum markers that can report on gene expression with a blood draw. These synthetic markers have well-defined parameters and thus form a useful model for understanding the maximum theoretical temporal resolution and precision of monitoring gene expression. These two variables describe, respectively, the fastest changes in gene expression and how accurately the kinetics can be reflected through a serum marker measurement. Developing more robust reporter systems requires understanding how biological properties of these markers, such as serum half-life or measurement noise, affect their kinetics. Here, we developed computational models describing in vivo behavior of synthetic serum markers to identify the most critical parameters for optimization. We show that serum half-life and measurement noise are the main contributors to the temporal precision and resolution of monitoring. Additionally, we demonstrate the ability to predict marker levels in various contexts, including constitutive and drug-induced expression.

bioengineering↗

bioRxiv2025

Synthetic Serum Markers Enable Noninvasive Monitoring of Gene Expression in Primate Brains

We demonstrate a noninvasive approach to measure transgene expression in the brain of nonhuman primates using blood tests with engineered reporters called Released Markers of Activity (RMAs). RMAs can exit the brain and enter the bloodstream via reverse-transcytosis across the blood-brain barrier. We demonstrate that these reporters can be used to repeatedly monitor expression of multiple transgenes in cortical and subcortical brain regions simultaneously over a period of three months. RMAs are also sensitive enough to detect circuit-specific Cre-dependent AAV expression. Through this study, the RMA platform provides a cost-efficient, noninvasive tool for neuroscience study of large animals, enabling sensitive, multiplexed, and repeatable measurements of gene expression in the brain with a blood test.

neuroscience↗

bioRxiv2025

Noninvasive Control of Seizure Threshold with Acoustically Targeted Chemogenetics

Many neurological and psychiatric diseases are characterized by pathological neuronal activity. Current treatments involve drugs, surgeries, and implantable devices to modulate or remove the affected region. However, none of these methods can be simultaneously non-invasive and possess site- and cell type specificity. Here, we apply a non-invasive neuromodulation approach called Acoustically-Targeted Chemogenetics, or ATAC, to increase the seizure threshold. Here, the ATAC approach used a multi-point focused ultrasound to transiently open the blood-brain barrier of the whole hippocampus and transduce pyramidal neurons with engineered G-protein-coupled receptors to inhibit their activity. To express the engineered receptors in the mouse hippo-campus, we used a recently engineered viral vector optimized for ultrasound-based gene delivery to the brain, AAV.FUS.3. In a mouse flurothyl seizure model, we showed successful gene delivery throughout the hippocampus, a significant neuronal activity inhibition as evidence by an increase in seizure threshold. Finally, we benchmarked these effects against a clinically prescribed drug that acts without spatial precision.

bioengineering↗

bioRxiv2025

Erasable Synthetic Serum Markers

Gene expression in the brain is typically evaluated using invasive biopsy or postmortem histology. Serum markers provide an alternative way to monitor the brain, but relatively few such markers exist. Additionally, the origin of serum markers often cannot be localized to a specific cell population, and monitoring dynamic changes in their gene expression is compromised by the same factor that makes the markers detectable - long serum half-life. Here we propose a paradigm to improve the sensitivity of serum marker measurement by modifying the markers in vivo, called erasable serum markers, or ESM. As a proof of concept, we use a well-controlled system with known half-life and tunable serum levels. This system, released markers of activity, or RMAs enable measurement of transgene expression in the brain through a simple blood test. RMAs are stable in blood, with a half-life of >100 h and can detect expression from as few as 12 neurons in mice. However, their long serum half-life also generates long-lasting background signals when RMA are used to track temporal changes in gene expression. By engineering on-demand erasable RMAs and injecting an intravenous targeted protease, we reduced RMA background signal by more than an order of magnitude without compromising the detection sensitivity. Similarly to previous RMA iteration, our approach showed a 65,000-fold increase in their signal over the baseline when expressed in a single brain region. Furthermore, we demonstrated that this erasable RMA system improves the dynamic range of detection for low-level promoter activity that is driven by physiological levels of c-Fos.

bioengineering↗

bioRxiv2025

Site-Specific Noninvasive Delivery of Retrograde Viral Vectors to the Brain

Neuronal activity underlies the brain function. Different behaviors, physiological processes, and disorders depend on which neurons are active at a given moment. Treating brain disorders without side effects will require exclusive control of disease-relevant neurons. Traditionally, small molecule drugs could control a subset of neurons that express a molecularly specific receptor. Local noninvasive therapies such as delivery of neuromodulatory agents with focused ultrasound blood-brain barrier opening (FUS-BBBO) also added spatial precision allowing one to control specific brain regions without surgery. However, the final characteristic of neurons, which other neurons they connect to, remains underexplored as a therapeutic target. If targeting neurons based on their connectivity was possible noninvasively, it would open the doors to broadly deployable precise therapies that can target selected subgroups of neurons within a brain region. Such delivery could be achieved with retrograde-tracing adeno-associated viral vectors (AAVs). For noninvasive delivery with FUS-BBBO, AAV9 has emerged as the most promising serotype. However, its retrograde-tracing version, the AAV9.retro, has not been evaluated for FUS-BBBO delivery. Here, we show that following such noninvasive delivery AAV9.retro can safely transduce neuronal projections with comparable efficiency to a direct intracranial injection. Compared to AAV8, a naturally occurring vector with low retrograde transduction, AAV9.retro offers superior retrograde transduction and comparable transduction at the site of delivery. Overall, we show that AAV9.retro is a valuable FUS-BBBO gene delivery vector, while also highlighting the surprising possibility of improved specificity of transduction of projections compared to invasive delivery.

bioengineering↗

bioRxiv2024

Monitoring in vivo transcription with synthetic serum markers

Understanding transcription profiles of living tissues is critical for biology and medicine. However, measurement of the transcript levels is typically done in homogenized tissues post-mortem. Here, we present a new platform that enables non-invasive monitoring of specific mRNA levels in vivo, without tissue destruction. We achieved this by combining two cutting-edge tools - synthetic serum markers, called Released Markers of Activity (RMAs), and RNA-based sensors of transcription. We call this platform IN-vivo Tracking of ACtive Transcription, or INTACT. In INTACT, when the target mRNA is expressed, the RNA sensor detects it and triggers the production and release of RMA reporters into the blood. Once in blood, the RMAs can be easily measured through a simple blood draw. Our data shows that INTACT can measure transcription of transgenes, as well as endogenous transcripts, such as c-Fos or Arc, both in vivo in the brain and in tissue culture. INTACT enables simple measurement of transcript level histories in genetically-targetable cell populations of living animals.

bioengineering↗

bioRxiv2023

Engineering Regionally-Activated Drugs for Neuroscience

The brain is comprised of multiple regions performing distinct functions. Within each of these regions, there are multiple cell types that can affect brain physiology. Finally, within each cell there are multiple signaling pathways, that, when activated or inhibited, control the cells activity, and consequently the brain function. For these reasons, methods that can control the brain with regional, cell-type, and molecular precision have been widely used in neuroscience. However, so far, achieving sustained control over a brain region with that level of specificity relied either on gene delivery or placement of invasive devices. While gene therapy holds great promise, the risks of genomic integration, vector toxicity, vector-directed immune response, high cost, and gene delivery to the brain pose significant challenges. On the other hand, invasive devices enable site-specific delivery of drugs but can also surgically damage the modulated brain region, carrying risks of infection and hemorrhage. Here, we present a new approach that can provide multi-day, noninvasive, site-specific control over specific cell types in the brain without the need to use invasive devices or gene delivery. To achieve this, we introduce a new paradigm called Regionally Activated Interstitial Drugs, or RAID, which delivers a protein-based catalytic centers, or RAID enzymes, to the brain using focused ultrasound blood-brain barrier opening. This catalytic center is designed to attach to the interstitial space in the brain where it remains for days after initial delivery. While the catalytic center is present in the brain, it can locally process an inert BBB permeable prodrug into an active drug, resulting in localized therapy. Our proof-of concept studies demonstrated that the engineered RAID enzymes can retain activity in the brain parenchyma for several days, allowing for noninvasive site-specific induction of neuronal activity that was sufficiently potent to elicit behavioral effects. Overall, the RAID paradigm enabled noninvasive, tunable, temporally-re-solved, site-specific, non-genetic, neuromodulation over multiple days. The RAID paradigm is versatile and can be applied to any enzyme and prodrug pair to control various aspects of central nervous system physiology.

bioengineering↗

bioRxiv2023

Acoustically-Targeted Measurement of Transgene Expression in the Brain

Gene expression is a critical component of brain physiology and activity, but monitoring this expression in the living brain represents a significant challenge. Here, we introduce a new paradigm called Recovery of Markers through InSonation (REMIS) for noninvasive measurement of gene expression in the brain with cell-type, spatial, and temporal specificity. Our approach relies on engineered protein markers that are designed to be expressed in neurons and exit into the interstitium. By applying ultrasound to targeted brain regions, these markers are released into the bloodstream, where they can be readily detected using biochemical techniques. REMIS can noninvasively confirm gene delivery and measure endogenous signaling in specific brain sites through a simple insonation and a subsequent blood test. Using REMIS, we successfully measured chemogenetic induction of neuronal activity in ultrasound-tar-geted brain regions. REMIS recovery of markers is reliable and demonstrated improved recovery of markers from the brain into the blood in every tested animal. Overall, our work establishes a noninvasive, spatially-specific means of monitoring gene delivery outcomes and endogenous signaling in mammalian brains, opening up possibilities for brain research and noninvasive monitoring of gene therapies in the brain.

bioengineering↗

bioRxiv2023

Acoustically Targeted Noninvasive Gene Therapy in Large Brain Regions

Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight, neuronal loss, or astrocyte activation. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, and found effective gene editing, but also a loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency and the first example of gene editing after site-specific noninvasive gene delivery to a large brain region.

bioengineering↗

bioRxiv2022

Engineered Serum Markers for Noninvasive Monitoring of Gene Expression in the Brain

Noninvasive efforts to map brain gene expression have been hampered by low sensitivity and limited access to the brain. Here, we introduce a new platform that enables multiplexed, noninvasive, and site-specific monitoring of brain gene expression through a novel class of engineered reporters called Released Markers of Activity (RMAs). Instead of detecting gene expression in the less accessible brain, RMA reporters exit from a known brain region into the blood, where they can be easily measured with biochemical techniques. Expressing RMAs at a single brain site, typically covering [~]1% of the brain volume, provides up to a 39,000-fold signal increase over the baseline in vivo. Further, expression of RMAs in as few as several hundred neurons was sufficient for their reliable detection. When placed under a promoter upregulated by neuronal activity, RMAs could be used to measure neuronal activity in specific brain regions with a simple blood draw. We found that chemogenetic activation of cells expressing Fos-responsive RMA increased serum levels of RMA over 4-fold compared to non-activated controls. By contrast, a control RMA expressed under a constitutive neuronal promoter did not show such upregulation, demonstrating multiplexed ratiometric measurement with RMAs and proving specificity of neuronal activity discrimination. Together, our study pioneers a new noninvasive paradigm for repeatable and multiplexed monitoring of gene expression in an intact brain with sensitivity that is currently unavailable through other noninvasive gene expression reporter systems.

neuroscience↗

bioRxiv2021

Engineering Viral Vectors for Acoustically Targeted Gene Delivery

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, limiting their scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency, requires ultrasound parameters close to tissue damage limits, and results in undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.

bioengineering↗

bioRxiv2018

Acoustically Targeted Chemogenetics for Noninvasive Control of Neural Circuits

Neurological and psychiatric diseases often involve the dysfunction of specific neural circuits in particular regions of the brain. Existing treatments, including drugs and implantable brain stimulators, aim to modulate the activity of these circuits, but are typically not cell type-specific, lack spatial targeting or require invasive procedures. Here, we introduce an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, called acoustically targeted chemogenetics, or ATAC, uses transient ultrasonic opening of the blood brain barrier to transduce neurons at specific locations in the brain with virally-encoded engineered G-protein-coupled receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. We demonstrate this concept in mice by using ATAC to noninvasively modify and subsequently activate or inhibit excitatory neurons within the hippocampus, showing that this enables pharmacological control of memory formation. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds, thereby overcoming some of the key limitations of conventional brain therapies.

neuroscience↗