Biology subjects

Wei, A. D.

Publications and source records attributed to Wei, A. D..

5 recordsLinked to original sources

bioRxiv2026

Optimizing Lentiviral Vector-Based Delivery of SCN1A transgenes to Mammalian Cells

The SCN1A gene encodes NaV1.1, a voltage-gated sodium channel protein that is necessary for neuronal excitability and whose loss-of-function mutations cause Dravet syndrome, a treatment-resistant childhood onset epilepsy. Gene replacement strategies for this syndrome are challenged by the large size of SCN1A and difficulty achieving stable cellular expression. Lentiviral vectors (LVVs) offer sufficient packaging capacity and genomic integration for defective SCN1A gene replacement. Here, we evaluated LVV-mediated delivery of different engineered SCN1A transgene sequences in human cells. LVV-transduced cells expressed full-length NaV1.1 protein that trafficked to the membrane and produced functional sodium currents. However, SCN1A transgene expression declined over time despite stable vector copy number, indicating post-integration regulatory limitations. Expression efficiency varied by SCN1A transgene sequence, with a codon-optimized variant showing higher expression despite lower LVV copy number. Treatment with sodium butyrate, a histone deacetylase inhibitor, significantly enhanced SCN1A transgene expression and partially rescued expression decay in a sequence-dependent manner. Incorporation of a ubiquitous chromatin opening element (UCOE) upstream of the promoter to maintain expression resulted in a trend of increased expression and increased responsiveness to butyrate. These findings demonstrate that sequence-specific and epigenetic factors may influence expression of large transgenes following lentiviral delivery, highlighting key challenges and design considerations for therapeutic SCN1A transgene expression.

synthetic biology↗

bioRxiv2025

Fentanyl blockade of K+ channels contribute to Wooden Chest Syndrome

Fentanyl is a potent synthetic opioid widely used perioperatively and illicitly as a drug of abuse 1,2. It is well established that fentanyl acts as a -opioid receptor agonist, signaling through Gi/o intracellular pathways to inhibit electrical excitability, resulting in analgesia and respiratory depression 3,4. However, fentanyl uniquely also triggers muscle rigidity, including respiratory muscles, hindering the ability to execute central respiratory commands or to receive external resuscitation. This potentially lethal condition is termed Wooden Chest Syndrome (WCS), the mechanisms of which are poorly understood 5-7. Here we show that fentanyl directly blocks a subset of EAG-class potassium channels 8. Our results also demonstrate that these channels are widely expressed in cervical spinal motoneurons, including those innervating the diaphragm. A significant fraction of these motoneurons is excited by fentanyl, concomitant with blockade of voltage-dependent non-inactivating K+ currents. In vivo electromyography revealed a persistent tonic component of diaphragmatic muscle activity elicited by fentanyl, but not morphine. Taken together our results identify a novel off-target mechanism for fentanyl action, independent of -opioid receptor activation, with a paradoxical excitatory effect that may underlie WCS. We anticipate these findings may inform the design of safer analgesics and generalize to other neuronal circuits implicated in fentanyl-related maladaptive behaviors.

neuroscience↗

bioRxiv2024

Mechanism of an animal toxin-antidote system

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

cell biology↗

bioRxiv2023

AAV-mediated interneuron-specific gene replacement for Dravet syndrome

Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy of SCN1A, which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV human SCN1A gene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form of SCN1A to circumvent AAV packaging limitations and deliver SCN1A via a dual vector approach using cell class-specific enhancers. These constructs produced full-length NaV1.1 protein and functional sodium channels in HEK293 cells and in brain cells in vivo. After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles of Scn1a, at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves of SCN1A, or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting of SCN1A conferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediated SCN1A gene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.

neuroscience↗

bioRxiv2020

Prior cocaine exposure increases firing to immediate reward while attenuating cue and context signals related to reward value in anterior insula

The insula contributes to behavioral control and is disrupted by substance abuse, yet we know little about the neural signals underlying these functions or how they are disrupted after chronic drug self-administration. Here, rats self-administered either cocaine (experimental group) or sucrose (control) for twelve consecutive days. After a one-month withdrawal period, we recorded from anterior insula while rats performed a previously learned reward-guided decision-making task. Cocaine-exposed rats were more sensitive to value manipulations and were faster to respond. These behavioral changes were accompanied by elevated counts of neurons in the insula that increased firing to reward. These neurons also fired more strongly at the start of long delay trials-when a more immediate reward would be expected, and fired less strongly in anticipation of the actual delivery of delayed rewards. Although reward-related firing to immediate reward was enhanced after cocaine self-administration, reward-predicting cue and context signals were attenuated. Significance StatementThe insula plays a clear role in drug-addiction and drug-induced impairments of decision-making, yet there is little understanding of its underlying neural signals. We found that chronic cocaine self-administration reduces cue and context-encoding in insula, while enhancing signals related to immediate reward. These changes in neural activity likely contribute to impaired decision-making and impulsivity observed after drug use.

neuroscience↗