Biology subjects

Chan, K. Y.

Publications and source records attributed to Chan, K. Y..

14 recordsLinked to original sources

bioRxiv2026

BI-TfR1 CapX rapidly transits the blood-brain barrier for efficient, low-dose gene delivery throughout the CNS

Safe, efficient gene delivery throughout the CNS remains a central obstacle to treating genetic diseases of the brain and spinal cord. We describe BI-TfR1 CapX, a human transferrin receptor (TfR1)-binding AAV capsid that, after a low intravenous dose in adult humanized TFRC mice, transduced more than 80% of cortical and spinal neurons and ~60% of cortical astrocytes, with reduced distribution and expression in several peripheral tissues relative to AAV9. Unlike other engineered BBB-crossing capsids, CapX bound the CNS vasculature within minutes and a large fraction of its genomes reached parenchymal nuclei within 24 hours. Delivering a Prnp-targeted epigenetic silencer (Prnp-CHARM), CapX reduced brain Prnp mRNA by over 95% requiring 18-fold fewer vector genomes per brain cell than AAV-PHP.eB for 50% lowering. A CapX delivered dual-vector cytosine base editor installed a protein-truncating PRNP stop codon lowering brain prion protein by 80%. These properties make CapX an attractive vehicle for human CNS gene therapy.

neuroscience↗

bioRxiv2026

Comparative analysis of microglia-targeted AAVs reveals capsid choice drives efficiency in vitro but has limited impact in vivo

Microglia play key roles in brain development, homeostasis, and neurodegeneration. Although multiple strategies for viral gene delivery to microglia have been reported, they have not been directly compared. Here, we developed microglia-targeting AAV capsids and benchmarked them against existing approaches. The novel capsids exhibit improved transduction efficiency in cultured mouse and human microglia, as well as neurons and astrocytes. However, when we compared microglial transduction efficiency of the novel capsids with published engineered and naturally occurring capsids after intracranial injection, all capsids achieved efficient and specific transduction when paired with a genome incorporating IBA1 promoter and miR-124 target sites. In contrast, CAG promoter did not support efficient microglial transduction. Moreover, blood-brain barrier- crossing capsids carrying IBA1 promoter and miR-124 target sites efficiently transduced microglia at high doses but exhibited off-target expression. Together, our work provides improved capsids for in vitro manipulation of microglia and establishes viral genome design, not capsid identity, as the principal determinant of efficient in vivo microglial targeting.

neuroscience↗

bioRxiv2026

Spinal circuits encode a map of the trunk to control skin twitches

Reflexive skin twitches are stereotypical behaviours, triggered in most mammals by mechanosensory stimuli. The neural circuits controlling this behaviour are thought to lie in the spinal cord, with motor neurons positioned in the lower cervical spinal cord innervating the cutaneous maximus muscle responsible for the twitches. The spatial matching of the motor output to the location of the sensory stimulus points to selective innervation of particular cutaneous maximus motor neurons by specific sensory-responsive circuits organised in a spatially-dependent manner. Using mouse genetics and viral tracing, we observed that a subset of dorso-lateral dI3 neurons forms ascending projections to motor neurons mediating skin twitches. Their projections are somatotopically organized to map a two dimensional space onto specific sub-compartments of the cutaneous maximus motor pool. Furthermore, direct optogenetic stimulation of thoraco-lumbar dI3 ascending projections in the cervical spinal cord induces skin twitches. Together, we demonstrate the circuit basis of a spinal sensory-motor representation of the dorsolateral trunk that participates in an ethological behaviour shared by most mammals allowing them to reduce the burdens of irritants such as insects.

neuroscience↗

bioRxiv2024

Clearance of protein aggregates during cell division

Protein aggregates are spatially organized and regulated in cells to prevent deleterious effects of proteostatic stress. Misfolding of proteins in the endoplasmic reticulum (ER) results in aggregate formation, but how the aggregates are processed especially during cell division is not well understood. Here, we induced proteostatic stress and protein aggregation using a proteostasis reporter, which is prone to misfolding and aggregation in the ER. Unexpectedly, we detected solid- like protein aggregates deposited mainly in the nucleus and surrounded by the ER membrane. The membrane-bound aggregates were then cleared as cells progressed through mitosis and cytokinesis. Aggregate clearance depended on Hsp70 family chaperones in the ER, particularly BiP, and proteasomal activity. The clearance culminated at mitotic exit and required cyclin-dependent kinase 1 (Cdk1) inactivation but was independent of the anaphase-promoting complex (APC/C). The ER reorganization that is active during mitosis and cytokinesis was required for the aggregate clearance. Thus, dividing cells reorganize the ER networks to allow BiP to clear the protein aggregates to maintain proteostasis in the newly divided cells.

cell biology↗

bioRxiv2023

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinsons disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.

neuroscience↗

bioRxiv2023

SC-Track: a robust cell tracking algorithm for generating accurate single cell linages from diverse cell segmentations

Computational analysis of fluorescent timelapse microscopy images at the single-cell level is a powerful approach to study cellular changes that dictate important cell fate decisions. Core to this approach is the need to generate reliable cell segmentations and classifications necessary for accurate quantitative analysis. Deep learning-based convolutional neural networks (CNNs) have emerged as a promising solution to these challenges. However, current CNNs are prone to produce noisy cell segmentations and classifications, which is a significant barrier to constructing accurate single-cell lineages. To address this, we developed a novel algorithm called Single Cell Track (SC-Track), which employs a hierarchical probabilistic cache cascade model based on biological observations of cell division and movement dynamics. Our results show that SC-Track performs better than a panel of publicly available cell trackers on a diverse set of cell segmentation types. This cell-tracking performance was achieved without any parameter adjustments, making SC-Track an excellent generalised algorithm that can maintain robust cell-tracking performance in varying cell segmentation qualities, cell morphological appearances and imaging conditions. Furthermore, SC-Track is equipped with a cell class correction function to improve the accuracy of cell classifications in multi-class cell segmentation time series. These features together make SC-Track a robust cell-tracking algorithm that works well with noisy cell instance segmentation and classification predictions from CNNs to generate accurate single-cell lineages and classifications.

bioinformatics↗

bioRxiv2022

Systematic multi-trait AAV capsid engineering for efficient gene delivery

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that evenly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 89% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited high production yields, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment, relative to AAV9, in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

synthetic biology↗

bioRxiv2022

Targeting AAV vectors to the CNS via de novo engineered capsid-receptor interactions

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of host cell macromolecules. Here, we screened for AAV capsids that bind two host cell proteins expressed on the mouse blood-brain barrier, LY6A or the related protein LY6C1. Introducing interactions with either protein target generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms. In contrast to the AAV-PHP.B capsid family, which interacts with LY6A and only exhibits its enhanced CNS tropism in a subset of mouse strains, the capsids that engage LY6C1 maintain their CNS tropism in BALB/cJ mice. Compared to conventional in vivo screens for CNS cell transducing capsids, a single round of protein target binding screening recovered significantly more capsids with enhanced performance that were validated in subsequent in vivo screens. Moreover, the initial screening round generated reproducible and quantitative target binding data that enabled the efficient machine learning-guided generation of more diverse targetspecific capsids. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.

synthetic biology↗

bioRxiv2022

Intravenous gene transfer throughout the brain of infant Old World primates using AAV

Adeno-associated viruses (AAVs) can enable robust and safe gene delivery to the mammalian central nervous system (CNS). While the scientific community has developed numerous neurotropic AAV variants for systemic gene-transfer to the rodent brain, there are few AAVs that efficiently access the CNS of higher order primates. We describe here AAV.CAP-Mac, an engineered AAV variant that enables systemic, brain-wide gene delivery in infants of two Old World primate species--the rhesus macaque (Macaca mulatta) and the green monkey (Chlorocebus sabaeus). We identified CAP-Mac using a multi-species selection strategy, initially screening our library in the adult common marmoset (Callithrix jacchus) and narrowing our pool of test-variants for another round of selection in infant macaques. In individual characterization, CAP-Mac robustly transduces human neurons in vitro and Old World primate neurons in vivo, where it targets all lobes of cortex, the cerebellum, and multiple subcortical regions of disease relevance. We use CAP-Mac for Brainbow-like multicolor labeling of macaque neurons throughout the brain, enabling morphological reconstruction of both medium spiny neurons and cortical pyramidal cells. Because of its broad distribution throughout the brain and high neuronal efficiency in infant Old World primates compared to AAV9, CAP-Mac shows promise for researchers and clinicians alike to unlock novel, noninvasive access to the brain for efficient gene transfer.

bioengineering↗

bioRxiv2021

pcnaDeep: A Fast and Robust Single-Cell Tracking Method Using Deep-Learning Mediated Cell Cycle Profiling

MotivationComputational methods that track single-cells and quantify fluorescent biosensors in time-lapse microscopy images have revolutionised our approach in studying the molecular control of cellular decisions. One barrier that limits the adoption of single-cell analysis in biomedical research is the lack of efficient methods to robustly track single-cells over cell division events. ResultsHere, we developed an application that automatically tracks and assigns mother-daughter relationships of single-cells. By incorporating cell cycle information from a well-established fluorescent cell cycle reporter, we associate mitosis relationships enabling high fidelity long-term single-cell tracking. This was achieved by integrating a deep-learning based fluorescent PCNA signal instance segmentation module with a cell tracking and cell cycle resolving pipeline. The application offers a user-friendly interface and extensible APIs for customized cell cycle analysis and manual correction for various imaging configurations. Availability and ImplementationpcnaDeep is an open-source Python application under the Apache 2.0 licence. The source code, documentation and tutorials are available at https://github.com/chan-labsite/PCNAdeep. Supplementary InformationSupplementary data are available online.

bioinformatics↗

bioRxiv2021

Targeted Neuronal Activation of the Gastrointestinal Tract Shapes the Environment of the Gut in Mice

The gastrointestinal (GI) tract is extensively innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia, which together regulate gut physiology. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Herein, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites, as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic migrating motor complexes and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally-activated ENS neurons differentially regulate the gut microbiome and GI physiology in mice, without involvement of signals from the brain.

physiology↗

bioRxiv2019

Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors useful for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of AAV-PHP.B vectors. Leveraging CNS tropism differences across mouse strains, we conducted a genome-wide association study, and rapidly identified and verified LY6A as an essential receptor for the AAV-PHP.B vectors in brain endothelial cells. Importantly, this newly discovered mode of AAV binding and transduction is independent of other known AAV receptors and can be imported into different cell types to confer enhanced transduction by the AAV-PHP.B vectors.

neuroscience↗

bioRxiv2018

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we developed a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We anatomically and functionally identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

neuroscience↗

bioRxiv2018

Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

neuroscience↗