RABID-seq Viral Barcode Tracing Single-Cell Interactions
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Solution Overview
Problem
Current methods for studying cell interactions in the central nervous system (CNS) face challenges due to technical complexity, throughput limitations, and a lack of single-cell resolution, making it difficult to investigate cell interactions and molecular mechanisms involved in astrocyte responses and CNS disorders like multiple sclerosis.
Innovation Solution
The development of RABID-seq, a method using G-deficient pseudorabies virus engineered to express a fluorescent mRNA-encoded barcode, allowing for the reconstruction of cellular interactions by encoding spatial relationships into the transcriptome, enabling the detection of cell interactions that would otherwise be missed by single-cell profiling alone.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell RNA-seq is used to profile individual cells, then single-cell resolution is achieved, but cell interactions cannot be detected
Solution Approach 1:
The patent uses rabies virus as an intermediary carrier to transfer barcode information between interacting cells. The virus infects a target cell and delivers a unique barcode, which then serves as a molecular tag indicating which cells have interacted with the target cell, thereby preserving interaction information that would otherwise be lost in single-cell profiling
Solution Approach 2:
The patent creates a copy of interaction information by using barcode sequences that are replicated and transferred through viral infection. Each interacting cell receives a copy of the target cell's barcode (or vice versa), creating a distributed record of cellular interactions that can be recovered and analyzed alongside single-cell transcriptome data
2Loss of information
If microdissection or photoactivatable markers are used to profile cell interactions, then interaction detection is enabled, but application to CNS and detection of small cell subsets is limited
Solution Approach 1:
The patent creates a universal tracing system that can be applied to any cell type in the CNS by using viral vectors that can infect diverse cell types. The rabies virus system is not limited to specific cell subsets or tissue types, making it versatile for studying interactions in the complex CNS environment and capable of detecting interactions involving rare cell populations
Solution Approach 2:
The system uses the natural biological processes of viral infection and RNA transcription to automatically trace cell interactions. The virus self-propagates through cell contacts and the barcode information is automatically recorded in the transcriptome of infected cells, eliminating the need for complex external marking or tracking systems
3Loss of information
If comprehensive cell interaction profiling is achieved, then molecular mechanisms are revealed, but technical complexity increases
Solution Approach 1:
The patent replaces complex mechanical or optical tracking systems with a molecular-based information encoding system. Instead of using physical markers or imaging systems to track cell interactions, the method uses viral RNA barcodes that are naturally incorporated into cell transcriptomes, allowing interaction data to be recovered through standard RNA sequencing workflows
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
RABID-seq provides a comprehensive and high-throughput approach to investigate cell interactions in the CNS with single-cell resolution, identifying novel interactions such as microglia-astrocyte interactions mediated by Sema4D-PlexinB2 signaling, which drives CNS pathology in autoimmune disorders like EAE and potentially multiple sclerosis.
Implementation Method 1
a method for identifying cell-cell contacts in a network of living cells comprising: (i) providing a network of living cells comprising rabies virus-infection-competent cells expressing a receptor for the foreign envelope protein and a functional rabies virus G protein
Implementation Method 2
a polynucleotide encoding: a fluorescent protein; a barcode sequence flanked by a first common sequence and a second common sequence
Data Source
AI summary
The present application relates to compositions and methods for tracing cell networks, e.g., for investigation of cell interactions in the CNS with single cell resolution.