Biology subjects

Engel, E. A.

Publications and source records attributed to Engel, E. A..

6 recordsLinked to original sources

bioRxiv2025

Administration of barcoded AAV capsid library to the putamen of non-human primates identifies variants with efficient retrograde transport

Adeno-associated viral vectors have become a leading choice for gene therapy in the central nervous system due to their safety profile, efficient neuronal transduction, and capacity for sustained transgene expression. We previously reported that AAV2-derived capsids developed using the BRAVE (Barcoded Rational AAV Vector Evolution) approach have enhanced retrograde transport properties in the rodent brain, compared to parental AAV2. Retrograde transport enables broader coverage of connected brain regions after a single focal intraparenchymal brain injection and is therefore a powerful tool for delivery of vectors to distant sites with potentially higher specificity, transduction efficacy and safety. Because transport properties can vary among species, we further characterized a barcoded library of 25 BRAVE-derived AAV2 capsid variants, along with the parental AAV2 serotype and benchmark AAV capsids, in brains of adult cynomolgus monkeys after intraputaminal dosing. Based on RNA and DNA amplicon sequencing, single-nucleus RNA sequencing, and histological assessment, we report here capsid variants with enhanced retrograde transport and expression compared to the parental AAV2 capsid. These properties make them potentially useful for disease indications in which broader brain coverage is desirable beyond the injection site.

neuroscience↗

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↗

bioRxiv2023

Neuronal expression of herpes simplex virus type-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation by activating Jun

Alpha herpesvirus (-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route, but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation, but also the factors that regulate the initial switch from productive to latent infection. Adeno associated virus (AAV) mediated expression of herpes simplex type 1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV transduced HSV VP16 specifically induced expression of proto-oncogenes including Jun and Pim2. In addition, HSV VP16 induces phosphorylation of Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.

microbiology↗

bioRxiv2022

The Alphaherpesvirus Latency Associated Promoter 2 (LAP2) Drives Strong Transgene Expression in Peripheral Tissue Depending on Administration Route and AAV Serotype

Adeno-associated virus (AAV) has shown great translational potential in treating a variety of diseases often requiring strong and ubiquitous transgene expression. However, the genetic payload of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are large in sizeable and susceptible to transcriptional silencing. We validated a short (404 bp), strong and persistent promoter obtained from the genome of pseudorabies virus (PRV) called alphaherpesvirus latency-associated promoter 2 (LAP2). We evaluated the biodistribution and potency of transgene expression in mouse peripheral tissue and organs when using AAV8-LAP2 and AAV9-LAP2, both of which achieved transgene expression like that of the ubiquitous promoter, EF1. LAP2 drives potent transgene expression in liver and kidney after systemic retro-orbital administration and in skeletal muscle after intramuscular delivery. Additionally, we observed broad transduction throughout the lung albeit at lower levels than other tissues. Notably, in skeletal muscle LAP2 resulted in preferential transduction of myofiber types 2. A direct side-by-side comparison between LAP2 and EF1, demonstrates that regardless of the AAV serotype and route of administration, LAP2 is as powerful and persistent as EF1 promoter despite being 66% smaller in size, thus allowing for larger therapeutic payloads.

cell biology↗

bioRxiv2020

Parallel organization of cerebellar pathways to sensorimotor, associative, and modulatory forebrain

Cerebellar outputs take polysynaptic routes to reach the rest of the brain, impeding conventional tracing. Here we quantify pathways between cerebellum and forebrain using transsynaptic tracing viruses and a whole-brain quantitative analysis pipeline. Retrograde tracing found a majority of descending paths originating from somatomotor cortex. Anterograde tracing of ascending paths encompassed most thalamic nuclei, especially ventral posteromedial, lateral posterior, mediodorsal, and reticular nuclei; in neocortex, sensorimotor regions contained the most labeled neurons, but higher densities were found in associative areas, including orbital, anterior cingulate, prelimbic, and infralimbic cortex. Patterns of ascending expression correlated with c-Fos expression after optogenetic inhibition of Purkinje cells. Our results reveal homologous networks linking single areas of cerebellar cortex to diverse forebrain targets. We conclude that shared areas of cerebellum are positioned to provide sensory-motor information to regions implicated in both movement and nonmotor function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/979153v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1bead0corg.highwire.dtl.DTLVardef@1531de2org.highwire.dtl.DTLVardef@1cf02f6org.highwire.dtl.DTLVardef@1cb6774_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

SMALL ALPHAHERPESVIRUS LATENCY-ASSOCIATED PROMOTERS DRIVE EFFICIENT AND LONG-TERM TRANSGENE EXPRESSION IN THE CENTRAL NERVOUS SYSTEM

Recombinant adeno-associated viral vectors (rAAV) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy, to identify three small latency-associated promoters (LAP) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404bp), LAP2 (498bp) and LAP1_2 (880bp). They drive chronic transcription of the virus encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAP in the CNS for six months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1 promoter (1264bp). Our data suggests that the LAP are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

neuroscience↗