Focused Ultrasound as a Novel Non-Invasive Method for the Delivery of Gold Nanoparticles to Retinal Ganglion Cells.

Purpose: The blood-retinal barrier (BRB) restricts the delivery of intravenous therapeutics to the retina, necessitating innovative approaches for treating retinal disorders. This study sought to explore the potential of focused ultrasound (FUS) to non-invasively deliver intravenously administered gold nanoparticles (AuNPs) across the BRB. FUS-BRB modulation can offer a novel method for targeted retinal therapy. Methods: AuNPs of different sizes and shapes were characterized, and FUS parameters were optimized to permeate the BRB without causing retinal damage in a rodent model. The delivery of 70-kDa dextran and AuNPs to the retinal ganglion cell (RGC) layer was visualized using confocal and two-photon microscopy, respectively. Histological and statistical analyses were conducted to assess the effectiveness and safety of the procedure. Results: FUS-BRB modulation resulted in the delivery of dextran and AuNPs to the RGC and inner nuclear layer. Smaller AuNPs reached the retinal layers to a greater extent than larger ones. The delivery of dextran and AuNPs across the BRB with FUS was achieved without significant retinal damage. Conclusions: This investigation provides the first evidence, to our knowledge, of FUS-mediated AuNP delivery across the BRB, establishing a foundation for a targeted and non-invasive approach to retinal treatment. The results contribute to developing promising non-invasive therapeutic strategies in ophthalmology to treat retinal diseases. Translational Relevance: Modifying the BRB with ultrasound offers a targeted and non-invasive delivery strategy of intravenous therapeutics to the retina.

Focused ultrasound gene delivery for the treatment of neurological disorders.

The transformative potential of gene therapy has been demonstrated in humans. However, there is an unmet need for non-invasive targeted gene delivery and regulation in the treatment of brain disorders. Transcranial focused ultrasound (FUS) has gained tremendous momentum to address these challenges. FUS non-invasively modulates brain cells and their environment, and is a powerful tool to facilitate gene delivery across the blood-brain barrier (BBB) with millimeter precision and promptly regulate transgene expression. This review highlights technical aspects of FUS-mediated gene therapies for the central nervous system (CNS) and lessons learned from discoveries in other organs. Understanding the possibilities and remaining obstacles of FUS-mediated gene therapy will be necessary to harness remarkable technologies and create life-changing treatments for neurological disorders.

The Patient as an Actor in His Care Pathway: Insights From the French Case.

In France, patients' right to take part in decisions regarding their health has been recognized by law since 2002. This legal recognition was the outcome of a long-standing call to allow all individuals to be "actors in their own health" and to co-develop their care pathway with the professionals involved. In practice, care pathways simultaneously intertwine both standardization and personalization dynamics, which involve different forms of professional-patient interaction. This article analyses the links between the organizational variables of care pathways, and the ways in which patients are involved in the management of their own pathway. To date, these links have received little attention in the management science and health literatures. We draw on material from a case study carried out in 2 French territories, combining the analysis of patient pathways with interviews conducted with professionals and carers. Building on this analysis, we propose a typology of patient profiles which distinguishes between their different forms of involvement in the development of their care pathway, based on its organizational characteristics.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

Anesthetic-loaded nanodroplets with focused ultrasound reduces agitation in Alzheimer's mice.

OBJECTIVE: Alzheimer's disease (AD) is often associated with neuropsychiatric symptoms, including agitation and aggressive behavior. These symptoms increase with disease severity, ranging from 10% in mild cognitive impairment to 50% in patients with moderate-to-severe AD, pose a great risk for self-injury and injury to caregivers, result in high rates of institutionalization and great suffering for patients and families. Current pharmacological therapies have limited efficacy and a high potential for severe side effects. Thus, there is a growing need to develop novel therapeutics tailored to safely and effectively reduce agitation and aggressive behavior in AD. Here, we investigate for the first time the use of focused ultrasound combined with anesthetic-loaded nanodroplets (nanoFUS) targeting the amygdala (key structure in the neurocircuitry of agitation) as a novel minimally invasive tool to modulate local neural activity and reduce agitation and aggressive behavior in the TgCRND8 AD transgenic mice. METHODS: Male and female animals were tested in the resident-intruder (i.e., aggressive behavior) and open-field tests (i.e., motor agitation) for baseline measures, followed by treatment with active- or sham-nanoFUS. Behavioral testing was then repeated after treatment. RESULTS: Active-nanoFUS neuromodulation reduced aggressive behavior and agitation in male mice, as compared to sham-treated controls. Treatment with active-nanoFUS increased the time male mice spent in social-non-aggressive behaviors. INTERPRETATION: Our results show that neuromodulation with active-nanoFUS may be a potential therapeutic tool for the treatment of neuropsychiatric symptoms, with special focus on agitation and aggressive behaviors. Further studies are necessary to establish cellular, molecular and long-term behavioral changes following treatment with nanoFUS.

Blood-brain barrier opening of the default mode network in Alzheimer's disease with magnetic resonance-guided focused ultrasound.

The blood-brain barrier (BBB) protects the brain but is also an important obstacle for the effective delivery of therapeutics in Alzheimer's disease and other neurodegenerative disorders. Transcranial magnetic resonance-guided focused ultrasound (MRgFUS) has been shown to reversibly disrupt the BBB. However, treatment of diffuse regions across the brain along with the effect on Alzheimer's disease relevant pathology need to be better characterized. This study is an open-labelled single-arm trial (NCT04118764) to investigate the feasibility of modulating BBB permeability in the default mode network and the impact on cognition, amyloid and tau pathology as well as BBB integrity. Nine participants [mean age 70.2 ± 7.2 years, mean Mini-Mental State Examination (MMSE) 21.9] underwent three biweekly procedures with follow-up visits up to 6 months. The BBB permeability of the bilateral hippocampi, anterior cingulate cortex and precuneus was transiently increased without grade 3 or higher adverse events. Participants did not experience worsening trajectory of cognitive decline (ADAS-cog11, MMSE). Whole brain vertex-based analysis of the 18F-florbetaben PET imaging demonstrated clusters of modest SUVR reduction in the right parahippocampal and inferior temporal lobe. However, CSF and blood biomarkers did not demonstrate any amelioration of Alzheimer's disease pathology (P-tau181, amyloid-ß42/40 ratio), nor did it show persistent BBB dysfunction (plasma PDGFRbeta and CSF-to-plasma albumin ratio). This study provides neuroimaging and fluid biomarker data to characterize the safety profile of MRgFUS BBB modulation in neurodegeneration as a potential strategy for enhanced therapeutic delivery.

Using focused ultrasound to modulate microglial structure and function.

Transcranial focused ultrasound (FUS) has the unique ability to target regions of the brain with high spatial precision, in a minimally invasive manner. Neuromodulation studies have shown that FUS can excite or inhibit neuronal activity, demonstrating its tremendous potential to improve the outcome of neurological diseases. Recent evidence has also shed light on the emerging promise that FUS has, with and without the use of intravenously injected microbubbles, in modulating the blood-brain barrier and the immune cells of the brain. As the resident immune cells of the central nervous system, microglia are at the forefront of the brain's maintenance and immune defense. Notably, microglia are highly dynamic and continuously survey the brain parenchyma by extending and retracting their processes. This surveillance activity aids microglia in performing key physiological functions required for brain activity and plasticity. In response to stressors, microglia rapidly alter their cellular and molecular profile to help facilitate a return to homeostasis. While the underlying mechanisms by which both FUS and FUS + microbubbles modify microglial structure and function remain largely unknown, several studies in adult mice have reported changes in the expression of the microglia/macrophage marker ionized calcium binding adaptor molecule 1, and in their phagocytosis, notably of protein aggregates, such as amyloid beta. In this review, we discuss the demonstrated and putative biological effects of FUS and FUS + microbubbles in modulating microglial activities, with an emphasis on the key cellular and molecular changes observed in vitro and in vivo across models of brain health and disease. Understanding how this innovative technology can modulate microglia paves the way for future therapeutic strategies aimed to promote beneficial physiological microglial roles, and prevent or treat maladaptive responses.

The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites.

Magnetic resonance imaging-guided focused ultrasound combined with microbubbles injected in the bloodstream (MRIgFUS) temporarily increases the permeability of the blood-brain barrier (BBB), which facilitates the entry of intravenously administered adeno-associated viruses (AAVs) from the blood to targeted brain areas. To date, the properties of the AAVs used for MRIgFUS delivery resulted in cell transduction limited to MRIgFUS-targeted sites. Considering future clinical applications, strategies are needed to deliver genes to multiple locations and large brain volumes while creating minimal BBB modulation. Here we combine MRIgFUS with a vector that has enhanced biodistribution following brain entry, AAV2-HBKO, to mediate broad gene delivery to targeted brain regions at levels with potential therapeutic relevance. Expression of a reporter gene was achieved in 13% and 21% of all neurons present in the striatum and thalamus, respectively, while targeting only 28% of the brain regions with MRIgFUS. Compared with AAV9, MRIgFUS-mediated delivery of AAV2-HBKO showed greater diffusion in the brain and a higher percentage of the neurons expressing the transgene. MRIgFUS AAV2-HBKO gene delivery to the brain has the potential to reach levels that are functionally and clinically relevant, and this even when using relatively low intravenous AAV dosages, compared with what is currently used in clinical trials.

Efficacy of gene delivery to the brain using AAV and ultrasound depends on serotypes and brain areas.

Focused ultrasound combined with intravenously injected microbubbles (FUS) is known to non-invasively, locally, and transiently increase the permeability of the blood-brain barrier (BBB). A promising approach for non-invasive gene delivery to the brain is to administer recombinant adeno-associated viruses (AAVs) intravenously and allow them to cross the BBB at precise FUS-targeted brain regions. FUS-AAV delivery has been achieved in animal models; however, the key elements influencing, guiding, and monitoring the success of FUS-AAV delivery to the brain remain largely unknown. We systematically compared the ability of AAV1, AAV2, AAV5, AAV8, AAV9, and AAVrg to enter four specific brain regions and transduce two main cell types: neurons and astrocytes. Our results demonstrate that the AAV serotype, the extent of FUS-induced BBB permeability, and the intrinsic properties of the targeted brain tissue influence the observed biodistribution, diffusion and transduction of AAV to cells of the cerebrovasculature and brain parenchyma. Non-invasive contrast-enhanced MR imaging was found to predict the efficacy of FUS-AAV delivery. Notably, we also found that AAVs with high biodistribution to peripheral organs result in low gene delivery to the brain when combined with FUS. Gene delivery by AAV1, AAV2, AAV5, AAV8 and AAV9 was highly and selectively localized to FUS-targeted brain areas. To obtain non-invasive gene delivery to multiple brain regions with one area of FUS-BBB modulation, we combined a modified AAV2 vector harboring enhanced retrograde transport properties (AAVrg) with FUS-mediated brain delivery. This allowed for gene delivery from the FUS-targeted site to multiple connected brain regions. This study demonstrates that MR imaging can be used as a non-invasive indication of AAV delivery to the brain, and that the properties of AAV serotypes influence the efficacy of gene delivery to the brain with FUS. AAVs that have minimal peripheral biodistribution are ideal candidates for enhanced, and perhaps exclusive with future serotypes, delivery to the brain with FUS. The characterization of parameters influencing FUS-AAV delivery to the brain are critical to the design of safe and efficient gene therapies, from preclinical studies to future clinical applications.

Sex differences in long-term fear and anxiety-like responses in a preclinical model of PTSD.

With a high prevalence of posttraumatic stress disorder (PTSD) in females, studying sex differences in preclinical models is of substantial importance. We have previously employed behavioural criteria to identify and characterize a subpopulation of rats that presented impaired fear extinction and long-term fear and anxiety responses following fear conditioning. We now exposed male and female rats to fear conditioning and extinction and segregated the animals into weak- (WE) and strong-extinction (SE) groups based on behavioural scores during extinction. Animals were subsequently tested for tone and context recall, as well as anxiety-like responses in the marble burying and novelty suppression of feeding (NSF) tests. Vaginal lavages were collected to characterize the phase of the estrous cycle during fear extinction. We found that females had reduced freezing during tone recall and a lower latency to feed in the NSF test. No differences were found in females undergoing extinction during high and low estrogen phases of the cycle in any of the performed tests. Overall, the percentage of animals that presented WE and SE phenotypes was similar in males and females. Both, WE males and females had increased freezing during tone and context recall. Along with our previous reports, WE males presented anxiety-like responses, particularly in the NSF compared to SE animals. In contrast, WE females buried less marbles than their SE mates. Future investigation including a larger number of behavioural tests are certainly required to corroborate our findings and ascertain potential mechanisms to explain the differences observed in our study.

Breached Barriers: A Scoping Review of Blood-Central Nervous System Barrier Pathology in Amyotrophic Lateral Sclerosis.

Introduction: Recent studies have implicated changes in the blood-central nervous system barriers (BCNSB) in amyotrophic lateral sclerosis (ALS). The objective of this scoping review is to synthesize the current evidence for BCNSB structure and functional abnormalities in ALS studies and propose how BCNSB pathology may impact therapeutic development. Methods: A literature search was conducted using Ovid Medline, EMBASE, and Web of Science, from inception to November 2021 and limited to entries in English language. Simplified search strategy included the terms ALS/motor neuron disease and [BCNSB or blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB)]. Henceforth, BCNSB is used as a term that is inclusive of the BBB and BSCB. Four independent reviewers conducted a title and abstract screening, hand-searched the reference lists of review papers, and performed a full text review of eligible studies. Included studies were original peer-reviewed full text publications, evaluating the structure and function of the BCNSB in preclinical models of ALS, clinical ALS, or postmortem human ALS tissue. There was no restriction on study design. The four reviewers independently extracted the data. Results: The search retrieved 2,221 non-duplicated articles and 48 original studies were included in the synthesis. There was evidence that the integrity of the BCNSB is disrupted throughout the course of the disease in rodent models, beginning prior to symptom onset and detectable neurodegeneration. Increased permeability, pharmacoresistance with upregulated efflux transporters, and morphological changes in the supporting cells of the BCNSB, including pericytes, astrocytes, and endothelial cells were observed in animal models. BCNSB abnormalities were also demonstrated in postmortem studies of ALS patients. Therapeutic interventions targeting BCNSB dysfunction were associated with improved motor neuron survival in animal models of ALS. Conclusion: BCNSB structural and functional abnormalities are likely implicated in ALS pathophysiology and may occur upstream to neurodegeneration. Promising therapeutic strategies targeting BCNSB dysfunction have been tested in animals and can be translated into ALS clinical trials.

MORPHIOUS: an unsupervised machine learning workflow to detect the activation of microglia and astrocytes.

BACKGROUND: In conditions of brain injury and degeneration, defining microglial and astrocytic activation using cellular markers alone remains a challenging task. We developed the MORPHIOUS software package, an unsupervised machine learning workflow which can learn the morphologies of non-activated astrocytes and microglia, and from this information, infer clusters of microglial and astrocytic activation in brain tissue. METHODS: MORPHIOUS combines a one-class support vector machine with the density-based spatial clustering of applications with noise (DBSCAN) algorithm to identify clusters of microglial and astrocytic activation. Here, activation was triggered by permeabilizing the blood-brain barrier (BBB) in the mouse hippocampus using focused ultrasound (FUS). At 7 day post-treatment, MORPHIOUS was applied to evaluate microglial and astrocytic activation in histological tissue. MORPHIOUS was further evaluated on hippocampal sections of TgCRND8 mice, a model of amyloidosis that is prone to microglial and astrocytic activation. RESULTS: MORPHIOUS defined two classes of microglia, termed focal and proximal, that are spatially adjacent to the activating stimulus. Focal and proximal microglia demonstrated activity-associated features, including increased levels of ionized calcium-binding adapter molecule 1 expression, enlarged soma size, and deramification. MORPHIOUS further identified clusters of astrocytes characterized by activity-related changes in glial fibrillary acidic protein expression and branching. To validate these classifications following FUS, co-localization with activation markers were assessed. Focal and proximal microglia co-localized with the transforming growth factor beta 1, while proximal astrocytes co-localized with Nestin. In TgCRND8 mice, microglial and astrocytic activation clusters were found to correlate with amyloid-ß plaque load. Thus, by only referencing control microglial and astrocytic morphologies, MORPHIOUS identified regions of interest corresponding to microglial and astrocytic activation. CONCLUSIONS: Overall, our algorithm is a reliable and sensitive method for characterizing microglial and astrocytic activation following FUS-induced BBB permeability and in animal models of neurodegeneration.

Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer's disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer's disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer's disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer's disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood-brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer's disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer's disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer's disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer's disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood-brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer's disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer's disease.

Transgene distribution and immune response after ultrasound delivery of rAAV9 and PHP.B to the brain in a mouse model of amyloidosis.

Efficient disease-modifying treatments for Alzheimer disease, the most common form of dementia, have yet to be established. Gene therapy has the potential to provide the long-term production of therapeutic in the brain following a single administration. However, the blood-brain barrier poses a challenge for gene delivery to the adult brain. We investigated the transduction efficiency and immunological response following non-invasive gene-delivery strategies to the brain of a mouse model of amyloidosis. Two emerging technologies enabling gene delivery across the blood-brain barrier were used to establish the minimal vector dosage required to reach the brain: (1) focused ultrasound combined with intravenous microbubbles, which increases the permeability of the blood-brain barrier at targeted sites and (2) the recombinant adeno-associated virus (rAAV)-based capsid named rAAV-PHP.B. We found that equal intravenous dosages of rAAV9 combined with focused ultrasound, or rAAV-PHP.B, were required for brain gene delivery. In contrast to rAAV9, focused ultrasound did not decrease the rAAV-PHP.B dosage required to transduce brain cells in a mouse model of amyloidosis. The non-invasive rAAV delivery to the brain using rAAV-PHP.B or rAAV9 with focused ultrasound triggered an immune reaction including major histocompatibility complex class II expression, complement system and microglial activation, and T cell infiltration.

Viral alpha-synuclein knockdown prevents spreading synucleinopathy.

The accumulation of aggregated alpha-synuclein (α-syn) in Parkinson's disease, dementia with Lewy bodies and multiple system atrophy is thought to involve a common prion-like mechanism, whereby misfolded α-syn provides a conformational template for further accumulation of pathological α-syn. We tested whether silencing α-syn gene expression could reduce native non-aggregated α-syn substrate and thereby disrupt the propagation of pathological α-syn initiated by seeding with synucleinopathy-affected mouse brain homogenates. Unilateral intracerebral injections of adeno-associated virus serotype-1 encoding microRNA targeting the α-syn gene reduced the extent and severity of both the α-syn pathology and motor deficits. Importantly, a moderate 50% reduction in α-syn was sufficient to prevent the spread of α-syn pathology to distal brain regions. Our study combines behavioural, immunohistochemical and biochemical data that strongly support α-syn knockdown gene therapy for synucleinopathies.

Intravenous and Non-invasive Drug Delivery to the Mouse Basal ForebrainUsing MRI-guided Focused Ultrasound.

Basal forebrain cholinergic neurons (BFCNs) regulate circuit dynamics underlying cognitive processing, including attention, memory, and cognitive flexibility. In Alzheimer's disease and related neurodegenerative conditions, the degeneration of BFCNs has long been considered a key player in cognitive decline. The cholinergic system thus represents a key therapeutic target. A long-standing obstacle for the development of effective cholinergic-based therapies is not only the production of biologically active compounds but also a platform for safe and efficient drug delivery to the basal forebrain. The blood-brain barrier (BBB) presents a significant challenge for drug delivery to the brain, excluding approximately 98% of small-molecule biologics and nearly 100% of large-molecule therapeutic agents from entry into the brain parenchyma. Current modalities to achieve effective drug delivery to deep brain structures, such as the basal forebrain, are particularly limited. Direct intracranial injection via a needle or catheter carries risks associated with invasive neurosurgery. Intra-arterial injection of hyperosmotic solutions or therapeutics modified to penetrate the BBB using endogenous transport systems lack regional specificity, which may not always be desirable. Intranasal, intrathecal, and intraventricular administration have limited drug distribution beyond the brain surface. Here, we present a protocol for non-invasively, locally, and transiently increasing BBB permeability using MRI-guided focused ultrasound (MRIgFUS) in the murine basal forebrain for delivery of therapeutic agents targeting the cholinergic system. Ongoing work in preclinical models and clinical trials supports the safety and feasibility of MRIgFUS-mediated BBB modulation as a promising drug delivery modality for the treatment of debilitating neurological diseases.

MR-guided focused ultrasound liquid biopsy enriches circulating biomarkers in patients with brain tumors.

BACKGROUND: Liquid biopsy is promising for early detection, monitoring of response, and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood from brain tumors, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized that MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy. METHODS: Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening (BBBO) alone (NCT03739905). RESULTS: Brain regions averaging 7.8 ± 6.0 cm3 (range 0.8-23.1 cm3) were successfully treated within 111 ± 39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6 ± 1.2-fold, P < .01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2 ± 1.9-fold, P < .01), and brain-specific protein S100b (1.4 ± 0.2-fold, P < .01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease- and post-BBBO-specific, in keeping with our hypothesis. We also found cfDNA-mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbor the tumor mutation. CONCLUSIONS: This first-in-human proof-of-concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.

Vasculotide restores the blood-brain barrier after focused ultrasound-induced permeability in a mouse model of Alzheimer's disease.

Focused ultrasound (FUS) is used to locally and transiently induce blood-brain barrier (BBB) permeability, allowing targeted drug delivery to the brain. The purpose of the current study is to evaluate the potential of Vasculotide to accelerate the recovery of the BBB following FUS disruption in the TgCRND8 mouse model of amyloidosis, characteristic of Alzheimer's disease (AD). Accelerating the restoration of the BBB post-FUS would represent an additional safety procedure, which could be beneficial for clinical applications. Methods: TgCRND8 mice and their non-transgenic littermates were treated with Vasculotide (250 ng, intraperitoneal) every 48 hours for 3 months. BBB permeability was induced using FUS, in presence of intravenously injected microbubbles, in TgCRND8 and non-transgenic mice, and confirmed at time 0 by MRI enhancement using the contrast agent gadolinium. BBB closure was assessed at 6, 12 and 20 hours by MRI. In a separate cohort of animals, BBB closure was assessed at 24-hours post-FUS using Evans blue injected intravenously and followed by histological evaluation. Results: Chronic Vasculotide administration significantly reduces the ultra-harmonic threshold required for FUS-induced BBB permeability in the TgCRND8 mice. In addition, Vasculotide treatment led to a faster restoration of the BBB following FUS in TgCRND8 mice. BBB closure after FUS is not significantly different between TgCRND8 and non-transgenic mice. BBB permeability was assessed by gadolinium up to 20-hours post-FUS, demonstrating 87% closure in Vasculotide treated TgCRND8 mice, as opposed to 52% in PBS treated TgCRND8 mice, 58% in PBS treated non-transgenic mice, and 74% in Vasculotide treated non-transgenic mice. In both TgCRND8 mice and non-transgenic littermates the BBB was impermeable to Evans blue dye at 24-hours post-FUS. Conclusion: Vasculotide reduces the pressure required for microbubble ultra-harmonic onset for FUS-induced BBB permeability and it accelerates BBB restoration in a mouse model of amyloidosis, suggesting its potential clinical utility to promote vascular health, plasticity and repair in AD.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.