Biology subjects

Mich, J. K.

Publications and source records attributed to Mich, J. K..

11 recordsLinked to original sources

bioRxiv2026

A Cross-Species Enhancer-AAV Toolkit for Cell Type-Specific Targeting Across the Basal Ganglia

The mammalian basal ganglia (BG) orchestrate motor, cognitive, and affective functions, yet cell type-specific genetic access remains limited, especially beyond rodents. Key structures implicated in movement and psychiatric disorders, including pallidum, subthalamic nucleus, and dopaminergic midbrain, lack scalable tools for cross-species targeting. Here, we present a comprehensive enhancer-AAV library enabling selective labeling and manipulation of major BG neuronal populations: striatal projection neuron subtypes, pallidal and subthalamic neurons, and midbrain dopaminergic and GABAergic populations. Using an evolutionarily informed discovery pipeline, we identified enhancers targeting canonical, non-canonical, and disease-relevant cell types, with validation demonstrating robust cross-species conservation of specificity between mouse and macaque. Computational modeling revealed sequence features predictive of in vivo performance, including motif grammar, chromatin accessibility, and evolutionary conservation, and identified distinct regulatory architectures across glial, projection, and interneuron lineages. This work establishes a comprehensive cross-species viral toolkit for the BG, unlocking previously inaccessible cell types for circuit dissection.

neuroscience↗

bioRxiv2026

Enhancer-based AAV approach for selective AADC delivery reduces motor symptoms and dyskinesia in Parkinson's mouse models

Degeneration of midbrain dopamine (DA) neurons and the resulting loss of striatal dopamine signaling are hallmarks of Parkinsons disease (PD). Although the dopamine precursor levodopa (L-DOPA) provides symptomatic relief, prolonged treatment often leads to abnormal involuntary movements (dyskinesia). Previous adeno-associated virus (AAV) approaches delivering aromatic L-amino acid decarboxylase (AADC) to the striatum under a ubiquitous promoter enhanced local dopamine synthesis and improved PD motor deficits, but this broad targeting strategy limited insight into the cellular populations underlying the behavioral improvements. Here, we engineered enhancer-driven AAVs to direct AADC expression to defined striatal and midbrain cell populations and paired these regulatory elements with a blood-brain barrier-penetrant capsid to enable both systemic and direct delivery. Targeted expression restored motor performance at reduced L-DOPA doses and decreased dyskinesia-like behaviors, producing improvements comparable to or greater than those achieved with ubiquitous expression. Distinct neuronal and non-neuronal populations each supported motor rescue but improved different behavioral domains to different extents, indicating complementary cell type-specific roles within PD-relevant circuits. Together, these findings establish enhancer-driven, cell type-specific AADC delivery as a better-tolerated strategy that enables rescue of motor deficits at lower L-DOPA doses in PD models.

neuroscience↗

bioRxiv2025

Circuit specific specialization of human basal ganglia astrocytes

Astrocytes shape synapses and circuits, yet human basal ganglia astrocyte diversity is incompletely defined. We built a multimodal atlas by integrating single-nucleus RNA-sequencing and chromatin accessibility with DNA methylation, 3D chromatin conformation, and spatial transcriptomics, then mapped basal ganglia programs onto a whole-brain reference. Astrocytes segregated into three anatomical subgroups spanning striatal gray matter, extra-striatal gray matter, and white matter, with subgroup-biased neurotransmitter transporters and synapse-associated programs consistent with differences in dominant afferent input. Within striatum, dorsal and ventral astrocyte populations aligned with distinct microcircuits and were conserved in nonhuman primates. A deep learning sequence model identified subgroup-associated enhancer code and, when benchmarked against published enhancer-AAV datasets, supported the design of candidate viral tools to target basal ganglia astrocyte programs in vivo. Together, these data define major axes of human astrocyte specialization and provide a framework for cell type-specific dissection of basal ganglia function.

neuroscience↗

bioRxiv2025

Technical and biological sources of noise confound multiplexed enhancer AAV screening

Cis-acting regulatory enhancer elements are valuable tools for gaining cell type-specific genetic access. Leveraging large chromatin accessibility atlases, putative enhancer sequences can be identified and deployed in adeno-associated virus (AAV) delivery platforms. However, a significant bottleneck in enhancer AAV discovery is charting their detailed expression patterns in vivo, a process that currently requires gold-standard one-by-one testing. Here we present a barcoded multiplex strategy for screening enhancer AAVs at cell type resolution using single cell RNA sequencing and taxonomy mapping. We executed a proof-of-concept study using a small pool of validated enhancer AAVs expressing in a variety of neuronal and non-neuronal cell types across the mouse brain. Unexpectedly, we encountered substantial technical and biological noise including chimeric packaging products, necessitating development of novel techniques to accurately deconvolve enhancer expression patterns. These results underscore the need for improved methods to mitigate noise and highlight the complexity of enhancer AAV biology in vivo.

neuroscience↗

bioRxiv2025

Extensive length and homology dependent chimerism in pool-packaged AAV libraries

Adeno-associated viruses (AAVs) have emerged as the foremost gene therapy delivery vehicles due to their versatility, durability, and safety profile. Here we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex AAV libraries that are packaged as a pool. The observed chimerism is length- and homology-dependent but capsid-independent, in some cases affecting the majority of packaged AAV genomes. These results have implications for the design and deployment of functional AAV libraries in both research and clinical settings.

genomics↗

bioRxiv2024

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for identifying functional enhancers in mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian cortex and to designing more efficient genetic tools to target cortical cell types.

genomics↗

bioRxiv2024

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

molecular 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↗

bioRxiv2023

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have also revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and the astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity across transgenes and in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

neuroscience↗

bioRxiv2023

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

bioengineering↗

bioRxiv2019

Epigenetic landscape and AAV targeting of human neocortical cell classes

Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes.

neuroscience↗