In vivo enhancer activity profiling platform for neuroinflammation research and gene therapy vector design

Overview

This research offering leverages an established massively parallel enhancer screening platform based on AAV-STARRseq technology to profile changes in gene regulatory activity during neuroinflammation in the mouse brain. The approach screens tens to hundreds of thousands of genomic fragments in vivo to identify enhancers whose activity changes in specific cell types and states during the inflammatory process. Beyond advancing understanding of neuroinflammatory disease mechanisms, the platform yields validated regulatory elements that can be engineered into cell-type and state-specific promoters for use in gene therapy vectors, enabling precise targeting of therapeutic genes to relevant cell populations.

Technical specifications

Core methodology:

• AAV-STARRseq-based in vivo enhancer screening – a self-transcribing approach where genomic fragments are cloned downstream of a minimal promoter, packaged into AAV capsids, and delivered stereotaxically into the mouse brain. Fragments with enhancer activity drive their own transcription and are quantified via next-generation sequencing.
• High-throughput scale – screens ten- to hundred-thousands of genomic sequences simultaneously, far exceeding conventional barcode-based strategies.
• Differential screening design – comparison of enhancer activity profiles between an inducible neuroinflammation mouse model and healthy controls at multiple time points.

Validation pipeline:

• Independent validation of identified hits by driving fluorescent reporter expression with the enhancer placed upstream of a minimal promoter.
• Histological colocalization analyses with cell-type marker proteins and projection markers to confirm cell-type specificity.
• FACS sorting of fluorescently labeled nuclei followed by single-nucleus sequencing to characterize targeted cell populations.
• Short (~500 bp) genomic sequences are used as candidate regulatory elements.

Key advantages:

• Provides functional readout of regulatory element activity, complementing conventional gene expression profiling.
• Identifies enhancers with specific activity profiles suitable for constructing engineered promoters in viral vectors.
• Applicable across multiple organs, not limited to brain tissue.

Technology readiness level

The core AAV-STARRseq methodology has been validated and published, with successful application demonstrated in the mouse brain and other organs. Individual genomic sequences identified through prior screens have been independently validated using fluorescent reporters, confirming cell-type-specific expression patterns. The proposed differential screening in a neuroinflammation model represents an extension of this validated platform into a disease-specific context. Hit validation through reporter constructs, histological analysis, and single-nucleus sequencing is built into the project plan. The resulting enhancer candidates and engineered promoters represent translational tools that could support future gene therapy vector development, though clinical application would require additional preclinical validation steps.