Biology subjects

Konofagou, E. E.

Publications and source records attributed to Konofagou, E. E..

8 recordsLinked to original sources

bioRxiv2025

Portable transcranial therapeutic ultrasound enhances targeted gene delivery for Parkinson's disease: from rodent models to non-human primates

Gene therapy for neurodegenerative diseases faces significant challenges due to the blood-brain barrier (BBB), which limits drug delivery to the central nervous system (CNS). While clinical trials for Parkinsons disease (PD) have progressed, administration of vectors expressing enzymatic or neurotrophic factor transgenes have required extensive optimization of the delivery method to achieve potentially therapeutic levels of transgene expression. Focused ultrasound (FUS) combined with microbubbles has emerged as a promising non-invasive strategy to transiently open the BBB for targeted gene delivery via viral nanocarriers including recombinant adeno-associated viruses (AAVs). However, key factors influencing FUS-mediated AAV delivery, including dose distribution and therapeutic efficacy, remain underexplored in non-human primates (NHPs). Here, we evaluated the feasibility of AAV9-CAG-GFP delivery using two portable therapeutic ultrasound modalities: ultrasound-guided, spherically-focused FUS (USgFUS) and a novel low-frequency linear array configuration for imaging and therapy called theranostic ultrasound (ThUS). In mice, FUS-sonicated regions exhibited a 25-fold increase in AAV9 biodistribution compared to systemic injection alone. Extending this approach to NHPs, we observed up to a 200-fold increase in AAV9 DNA in treated brain regions, including PD-relevant structures. In assessing the translational therapeutic potential of this technique, ThUS-mediated AAV9-hSyn-hNTRN (human neurturin) delivery in a toxin mouse model of PD facilitated the rescue of up to 80% and 75% of degenerated dopaminergic neurons in the substantia nigra and striatum, respectively. These findings demonstrate that portable ultrasound technologies can non-invasively enhance AAV9 delivery to targeted brain regions in both mice and NHPs relative to what can be achieved with intravenous (IV) delivery of the same capsid alone. With further development, these approaches may offer a clinically viable, non-invasive alternative for gene therapy in neurodegenerative diseases. One sentence summaryBBB opening with portable therapeutic ultrasound non-invasively increased viral gene delivery to the brain after systemic AAV vector administration in mice and rhesus macaques.

bioengineering↗

bioRxiv2025

Safe focused ultrasound-mediated blood-brain barrier opening is driven primarily by transient reorganization of tight junctions

Focused ultrasound (FUS) with microbubbles opens the blood-brain barrier (BBB) to allow targeted drug delivery into the brain. The mechanisms by which endothelial cells (ECs) respond to either low acoustic pressures known to open the BBB transiently, or high acoustic pressures that cause brain damage, remain incompletely characterized. Here, we use a mouse strain where tight junctions between ECs are labelled with eGFP and apply FUS at low (450 kPa) and high (750 kPa) acoustic pressures, after which mice are sacrificed at 1 or 72 hours. We find that the EC response leading to FUS-mediated BBB opening at low pressures is localized primarily in arterioles and capillaries, and characterized by a transient loss and reorganization of tight junctions. BBB opening still occurs at low safe pressures in mice lacking caveolae, suggesting that it is driven primarily by transient dismantlement and reorganization of tight junctions. In contrast, BBB opening at high pressures is associated with obliteration of EC tight junctions that remain unrepaired even after 72 hours, allowing continuous fibrinogen passage and persistent microglial activation. Single-cell RNA-sequencing of arteriole, capillary and venule ECs from FUS mice reveals that the transcriptomic responses of ECs exposed to high pressure are dominated by genes belonging to the stress response and cell junction disassembly at both 1 and 72 hours, while lower pressures induce primarily genes responsible for intracellular repair responses in ECs. Our findings suggest that at low pressures transient reorganization of tight junctions and repair responses mediate safe BBB opening for therapeutic delivery. Significance StatementFocused ultrasound with microbubbles is used as a noninvasive method to safely open the BBB at low acoustic pressures for therapeutic delivery into the CNS, but the mechanisms mediating this process remain unclear. Kugelman et al., demonstrate that FUS-mediated BBB opening at low pressures occurs primarily in arterioles and capillaries due to transient reorganization of tight junctions. BBB opening still occurs at low safe pressures in mice lacking caveolae, suggesting a transcellular route-independent mechanism. At high unsafe pressures, cell junctions are obliterated and remain unrepaired even after 72 hours, allowing fibrinogen passage and persistent microglial activation. Single-cell RNA-sequencing supports cell biological findings that safe, FUS-mediated BBB opening may be driven by transient reorganization and repair of EC tight junctions.

bioengineering↗

bioRxiv2025

Characterization of Microbubble Cavitation in Theranostic Ultrasound-mediated Blood-Brain Barrier Opening and Gene Delivery

RationaleThe characterization of microbubble activity has proven critical in assessing the safety and efficacy of ultrasound-mediated blood-brain barrier (BBB) opening and drug and gene delivery. In this study, we build upon our previous work on theranostic ultrasound (ThUS)-mediated BBB opening (ThUS-BBBO) and conduct for the first time a comprehensive characterization of the role of microbubble cavitation in ThUS-BBBO, as well as its impact on gene delivery with adeno-associated viruses (AAV). MethodsA repurposed imaging phased array was used throughout the study to generate focused transmits and record microbubble activity through high-resolution power cavitation imaging (PCI). The cavitation of microbubbles under ThUS pulses was first characterized in flow phantom using pulse lengths ranging from 1.5 to 20 cycles and under varying microbubble flow rates using a separate single-element transducer a passive cavitation detector (PCD). A comprehensive in vivo study in mice was then conducted to characterize the in vivo microbubble activity under ThUS and correlate the resulting cavitation with AAV-mediated transgene delivery and expression. The transcranial microbubble activity was first detected in two mice using a PCD, to confirm the findings of the flow phantom study. Next, three mouse studies were conducted to evaluate the relationship between cavitation and AAV delivery; one with three different microbubble size distributions using polydisperse and size-isolated microbubbles, one with variable burst length and burst repetition frequency, and one with different AAV serotypes and injection doses. Electronic beam steering was used to induce bilateral BBB opening with 1.5 cycle on the left and 10 cycles on the right hemisphere. Cavitation dose was correlated with BBB opening volume, AAV transgene expression was evaluated with immunofluorescence staining and histological safety was assessed with T2* imaging and Hematoxylin and Eosin staining. ResultsFrequency domain analysis in the phantoms revealed a broadband-cavitation dominance at the shorter pulse lengths, while harmonic cavitation components are significantly increased for longer pulses. The PCD was better at detecting higher frequency harmonics, while the signal received by the theranostic array was more broadband dominated. Analysis of signals in the time domain showed that the longer pulses induce higher microbubble collapse compared to short pulses. In the transcranial in vivo experiments, the PCD was able to detect increased harmonic cavitation for 10-cycle pulses. The microbubble study showed that 3-5 m microbubbles resulted in the largest cavitation doses, BBBO volumes and AAV transgene expression compared to the smaller microbubble sizes. The burst sequence study revealed that the sequences with shorter bursts and faster burst repetition frequencies induce larger BBBO volumes and AAV transduction due to faster microbubble replenishment in the focal volume. Increased erythrocyte extravasation was observed on the hemisphere sonicated with 10-cycle pulses. Transgene expression was also increased with injection dose, without notable side effects during the three-week survival period. Finally, AAV9 was shown to be the serotype with the highest transduction efficiency compared to AAV2 and AAV5 at the same injected dose. ConclusionsThis is the first comprehensive study into the microbubble cavitation under theranostic ultrasound. The phantom and in vivo studies show that the mechanism of ThUS-BBBO is mainly transient cavitation dominant, as microbubble collapse increases with pulse length despite the increased harmonic frequency response. Increased cavitation dose resulted in larger BBBO volumes and transgene expression in vivo. While ThUS induced microhemorrhage for most of the studied conditions, it did not have an impact on the survival and behavior of the mice.

bioengineering↗

bioRxiv2024

Response of Serum-isolated Extracellular Vesicles to Focused Ultrasound Blood-Brain Barrier Opening

The blood-brain barrier (BBB) limits drug delivery to the brain and the movement of neurological biomarkers between the brain and blood. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) noninvasively opens the BBB, allowing increased molecular transport to and from the brain parenchyma. Despite being initially developed as a drug delivery method, FUS-BBBO has shown promise both as a neuroimmunotherapeutic modality, and as a way of improving neurological disease diagnosis via amplification of disease biomarker circulation. Recently, the role of extracellular vesicles (EVs) in modulating the neuroimmune system and in improving biomarker detection has sparked research interest. However, despite their potential role in modulating FUS-BBBO-induced neuroimmunotherapy and their ability to improve biomarker specificity after treatment, the EV response to FUS-BBBO had not been extensively characterized prior to this study. In this study, we investigated the effect of FUS-BBBO on EV concentration and content in the serum of mice and Alzheimers Disease (AD) patients. We observed a 164% increase in murine EV concentration one hour after treatment, as well as an increase in EV RNA associated with FUS-BBBO neuroimmunotherapy. Patient EV concentration also increased one hour after treatment and was dependent on the volume of BBB opening three days post-treatment. Furthermore, EV isolation was found to significantly enhance the amplification of AD biomarker detection by FUS-BBBO. Overall, we present the first evidence of altered murine and AD patient EV concentration and content in response to FUS-BBBO, providing evidence of EVs role within FUS-BBBO neuroimmunotherapy as well as their utility in improving FUS-BBBO biomarker amplification.

bioengineering↗

bioRxiv2024

Transcranial Functional Ultrasound Imaging Detects Focused Ultrasound Neuromodulation Induced Hemodynamic Changes in Mouse and Nonhuman Primate Brains In Vivo

BackgroundFocused ultrasound (FUS) is an emerging non-invasive technique for neuromodulation in the central nervous system (CNS). Functional ultrasound imaging (fUSI) leverages ultrafast Power Doppler Imaging (PDI) to detect changes in cerebral blood volume (CBV), which correlate well with neuronal activity and thus hold promise to monitor brain responses to FUS. ObjectiveInvestigate the immediate and short-term effects of transcranial FUS neuromodulation in the brain with fUSI by characterizing hemodynamic responses. MethodsWe designed a setup that aligns a FUS transducer with a linear array to allow immediate subsequent monitoring of the hemodynamic response with fUSI during and after FUS neuromodulation (FUS-fUSI) in lightly anesthetized mice. We investigated the effects of varying pressures and transducer positions on the hemodynamic responses. ResultsWe found that higher FUS pressures increase the size of the activated brain area, as well as the magnitude of change in CBV and could show that sham sonications did not produce hemodynamic responses. Unilateral sonications resulted in bilateral hemodynamic changes with a significantly stronger response on the ipsilateral side. FUS neuromodulation in mice with a cranial window showed distinct activation patterns that were frequency-dependent and different from the activation patterns observed in the transcranial model. ConclusionfUSI is hereby shown capable of transcranially monitoring online and short-term hemodynamic effects in the brain during and following FUS neuromodulation.

bioengineering↗

bioRxiv2023

Detection of Blood Volume Reduction and Vasoconstriction Following Focused Ultrasound Blood-Brain Barrier Opening Using Ultrasound Flow Imaging

Microbubble-mediated focused ultrasound (FUS) offers a non-invasive treatment for transient and localized blood-brain barrier (BBB) opening for drug delivery or immunostimulation. It is known that FUS-induced BBB opening is accompanied by blood flow changes, vasoconstriction, and vasodilation, as validated by optical microscopy through a cranial window. In this study, we introduce a novel method for quantifying vascular changes after FUS-induced BBB opening by employing ultrasound flow imaging in mice. We acquired pre-FUS and post-FUS ultrasound flow images with the same microbubble concentration in the brain. Contrast-enhanced power Doppler (CEPD) images and ultrasound localization microscopy images were obtained to evaluate changes in cerebral blood volume and vessel diameter at the sonicated region of the brain. Our findings demonstrate that FUS leads to a reduction in blood volume at the treated region, with vasoconstriction being more dominant than vasodilation. Furthermore, we show that transcranial CEPD can detect local blood reduction following FUS, which spatially coincides with the edema region identified in T2-weighted MRI. Our findings suggest that ultrasound flow imaging has the potential to serve as a cost-effective and immediate monitoring tool for evaluating the safety and efficacy of FUS-induced BBB opening.

bioengineering↗

bioRxiv2022

Focused ultrasound-mediated brain genome editing

Gene editing in the mammalian brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

bioengineering↗

bioRxiv2021

Long Term Study of Motivational and Cognitive Effects of Low-intensity Focused Ultrasound Neuromodulation in the Dorsal Striatum of Nonhuman Primates

Noninvasive brain stimulation using focused ultrasound (FUS) has many potential applications as a research and clinical tool, including incorporation into neural prosthetics for cognitive rehabilitation. To develop this technology, it is necessary to evaluate the safety and efficacy of FUS neuromodulation for specific brain targets and cognitive functions. It is also important to test whether repeated long-term application of FUS to deep brain targets improves or degrades behavioral and cognitive function. To this end, we investigated the effects of FUS in the dorsal striatum of nonhuman primates (NHP) performing a visual-motor decision-making task for small or large rewards. Over the course of 2 years, we performed 129 and 147 FUS applications, respectively, in two NHP. FUS (0.5 MHz @ 0.2 - 0.8 MPa) was applied to the putamen and caudate in both hemispheres to evaluate the effects on movement accuracy, motivation, decision accuracy, and response time. Sonicating the caudate or the putamen unilaterally resulted in modest but statistically significant improvements in motivation and decision accuracy, but at the cost of slower reaction times. The effects were dose (i.e., FUS pressure) and reward dependent. There was no effect on reaching accuracy, nor was there long-term behavioral impairment or neurological trauma evident on T1-weighted, T2-weighted, or susceptibility-weighted MRI scans. Sonication also resulted in significant changes in resting state functional connectivity between the caudate and multiple cortical regions. The results indicate that applying FUS to the dorsal striatum can positively impact the motivational and cognitive aspects of decision making. The capability of FUS to improve motivation and cognition in NHPs points to its therapeutic potential in treating a wide variety of human neural diseases, and warrants further development as a novel technique for non-invasive deep brain stimulation.

neuroscience↗