Browsing by Author "Szablowski, Jerzy"

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    Acoustically targeted measurement of transgene expression in the brain
    (2025-04-25) Seo, Joon Pyung; Szablowski, Jerzy
    Gene expression is a critical component of brain physiology and activity. Brain development, function, and plasticity relies on a regulated process of converting genetic information into functional products. However, monitoring gene expression in the living brain has been a significant challenge. The confined structure of the brain, protected by the cranium and shielded by the blood-brain barrier, has posed difficulty in non-invasive and sensitive measurement of gene expression with specificity. The aim of this thesis is to develop a new paradigm of technology capable of measuring gene expression in the brain non-invasively with cell-type, spatial, and temporal specificity. To achieve this, we combined focused ultrasound liquid biopsy and recovery of engineered protein markers that are designed to be expressed in neurons and exit into the brain’s interstitium. When ultrasound is applied to targeted brain regions, it temporarily opens the blood-brain barrier and releases the interstitial markers into the bloodstream. Once in blood, the markers can be readily detected from blood collection followed by compatible biochemical techniques. We call this Recovery of Markers through InSonation (REMIS). We demonstrated improved recovery of engineered Gaussia luciferase marker, under constitutive promoter, from the brain into the blood in every tested animal. Further, we implemented the markers to measure endogenous neuronal signaling activity by controlling the expression of the marker under a genetic circuit that responds to c-Fos when activated by enhanced neuronal activity. Lastly, we measured enhanced serum level of overexpressed human alpha-synuclein in the engineered Parkinson’s disease model mThy1-aSyn (Line61) mouse strain with REMIS. Overall, our work demonstrates the feasibility of combining engineered gene expression reporters and focused ultrasound liquid biopsy to noninvasively and specifically measure gene expression in the intact brain.
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    Engineered serum markers for non-invasive monitoring of gene expression in the brain
    (2024-08-05) Lee, Sangsin; Szablowski, Jerzy
    Gene expression provides the physiological underpinning for myriad biological processes, including those in the brain. However, monitoring the brain gene expression has been challenging due to the brain’s natural, confined architecture which restricts access to its thick, delicate tissue. Consequently, most existing approaches directly measure RNA abundance or transcriptional changes in an invasive manner, such as by histology or implanted optical devices, the result of which is irreversible physical damage to the brain. The aim of this thesis is to develop a new paradigm of molecular technology capable of measuring brain gene expression non-invasively, while ensuring high sensitivity, scalability, and precise spatial, temporal, and cellular resolution. As a first step towards this goal, we engineered a new class of synthetic, serum-based markers, called Released Markers of Activity (RMAs), that allows for non-invasive monitoring of gene expression with a simple blood test. Genetic tagging of specific genes of interest in the brain with RMA results in the release of RMA reporters from the brain into the bloodstream in response to the level of molecular activity of the gene. Once RMAs are deposited inside the blood, the blood samples can be analyzed using compatible biochemical techniques. Our initial readout method quantifies released RMAs in the blood by measuring luciferase activity, which provided high sensitivity with detectability down to approximately twelve labeled neurons in the mouse brain. Further, genetic tagging of RMAs to the neuronal activity-related gene Fos enabled the discrimination of brain activity through blood analysis. Because RMAs function as protein markers, this paradigm delivers greater scalability for measuring multiple genes or even enabling high-throughput gene analysis of the brain. The next devised strategy which combines the methods of nanopore protein sequencing and a machine learning-based classifier measures differentially barcoded RMAs for non-invasive, parallel readout of multiple genes in the brain. Taken together, our technology presents a novel and radical new reporter system for safe, repeatable, and multiplexed measurement of gene expression in an intact brain.
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    Recovery of Markers through Insonation: An alternative to monitoring gene expression in deep tissues
    (2023-02-13) Seo, Joon Pyung; Szablowski, Jerzy
    We developed a method to noninvasively measure transgene expression in the specific brain regions using a blood test. To achieve this, we used engineered protein reporters that are released from the cells into the brain interstitium. We then used focused ultrasound (FUS) to transiently open the blood-brain barrier (BBBO) and release these reporters into the blood. We call this approach REcovery of Markers through InSonation, or REMIS. We show that levels of markers secreted from neurons into the serum correlate with the levels of transgene expression in the brain. We measured up to 5.5-fold increase of marker levels in the blood after opening of 8% of the blood-brain barrier (BBB). We show the procedure is well tolerated and avoids significant tissue damage, consistent with other BBB opening studies. Finally, we show that the marker is released from the brain over prolonged periods of time. The levels of the released marker were comparable at 7.5 minutes and 2 hours after FUS-BBBO (p=0.31, paired t-test). At the same time, the serum half-life of the marker injected intravenously was 7.6 minutes. This suggests that the marker was released from the brain gradually, replenishing the marker in the serum over 2 hours, and indicating a broad time window available for marker collection. Monitoring gene expression in deep tissues of living animals is critical for in vivo studies and translation of gene therapies. This technology allows for site-specific measurement of gene expression in the brain.