WDFY3 Regulator Identification in Apoptotic Cell Uptake Screening
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Solution Overview
Problem
Current approaches lack an unbiased method for genome-wide screening of regulators involved in efferocytosis of apoptotic cells in mammalian cells, which is crucial for understanding and addressing impaired efferocytosis associated with various diseases.
Innovation Solution
A pooled genome-wide CRISPR knockout screen was established in primary murine bone marrow-derived macrophages to identify key regulators of efferocytosis, specifically uncovering WDFY3 (Alfy) as a novel regulator involved in the recognition and uptake of apoptotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hypothesis-driven approaches are used to identify regulators of efferocytosis, then known regulators can be successfully identified, but new regulators from diverse and unexpected gene classes cannot be discovered
Solution Approach 1:
The patent uses a pooled CRISPR knockout screen as an intermediary system that combines the precision of targeted gene disruption with the versatility of genome-wide screening. This approach allows systematic identification of both known and novel efferocytosis regulators by mediating between hypothesis-driven research and unbiased discovery
Solution Approach 2:
The screening platform established in the patent is designed to be universally applicable for identifying regulators of efferocytosis across different cell types and conditions. The pooled CRISPR approach enables simultaneous screening of multiple genes and functions, making the system multi-functional for both validation of known regulators and discovery of novel ones
2Productivity
If genome-wide CRISPR knockout screens are performed in immortalized or tumor-derived monocytic cell lines, then screening can be conducted, but physiological relevance to primary macrophages is lacking
Solution Approach 1:
The patent employs primary murine bone marrow-derived macrophages, which are short-lived primary cells rather than immortalized lines. These cells are cultured freshly for each experiment, accepting their limited lifespan in exchange for high physiological relevance and accurate representation of in vivo macrophage function in efferocytosis screening
3Ease of manufacture
If phagocytosis of beads is used to screen for efferocytosis regulators, then screening can be performed, but AC-specific recognition receptors and stiffness/size-dependent mechanisms cannot be recapitulated
Solution Approach 1:
The patent uses apoptotic cells as the actual substrate for efferocytosis screening rather than bead copies. This approach copies the physiological process accurately, allowing recognition of AC-specific receptors and mechanisms that depend on the actual physical and biochemical properties of dying cells, including their membrane stiffness and size
Solution Approach 2:
The screening system changes the key parameter of substrate properties from artificial beads to actual apoptotic cells. This parameter change enables the system to capture AC-specific recognition mechanisms, including receptor-ligand interactions and mechanical properties that are unique to dying cells and cannot be replicated by standardized beads
Data Source
AI summary
Provided here are methods to prevent, inhibit or treat an autoimmune disease or a vascular or cardiovascular disorder in a mammal, comprising administering to the mammal a composition comprising an effective amount of isolated nucleic acid comprising a nucleotide sequence encoding WDFY3/Alfy or a portion thereof, a nucleotide sequence comprising a WDFY3/Alfy-specific long non-coding RNA (lncRNA) or a corresponding DNA sequence, a nucleotide sequence comprising a portion of a lncRNA WDFY3-AS2 or a corresponding DNA sequence, or isolated WDFY3/Alfy or a portion thereof.