Compact FKBP13 Drug-Responsive Domain for AAV Payload Regulation
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Solution Overview
Problem
Current methods for regulating genetically modified cells or gene editing agents are limited by the capacity of viral or non-viral vectors, making it challenging to effectively regulate larger payloads, such as gene-editing agents, or transduction using smaller vectors like adeno-associated virus (AAV) vectors.
Innovation Solution
The development of a compact drug-responsive domain (DRD) based on FKBP13 or its variants, which can be operably linked to gene-editing payloads. This DRD is responsive to ligands such as FK506 and rapamycin, allowing for dose-dependent regulation of gene-editing activity by modulating the abundance and activity of the payload.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If known drug responsive domains are used to regulate payloads, then regulation capability is achieved, but the nucleic acid construct size increases beyond the capacity of selected viral or non-viral vectors
Solution Approach 1:
The patent extracts and utilizes only the essential functional portion of FKBP13 (amino acids 1-76 or 1-86) that contains the ligand-binding domain, eliminating unnecessary portions of the full-length protein. This extraction allows the DRD to maintain regulation capability while reducing the nucleic acid construct size to fit within AAV vector capacity.
Solution Approach 2:
The patent applies local quality by focusing on the specific functional region of FKBP13 (the ligand-binding domain) rather than using the entire protein. By optimizing this local region with specific mutations (e.g., M54A, D90G, M96V) that enhance ligand binding while reducing overall size, the construct achieves effective regulation within size constraints.
2Reliability
If larger payloads such as gene-editing agents are used, then therapeutic benefit is increased, but the ability to transduce using smaller vectors like AAV is compromised
Solution Approach 1:
The patent segments the gene-editing system into modular components (e.g., Cas9 protein, guide RNA, and DRD-regulated elements) that can be independently optimized and packaged. The compact DRD (amino acids 1-76 or 1-86 of FKBP13) is designed as a separate regulatable module that controls the expression or activity of the gene-editing payload, allowing the entire construct to fit within AAV packaging limits while maintaining full therapeutic functionality.
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
The use of compact FKBP13-based DRDs enables efficient regulation of gene-editing payloads, optimizing therapeutic benefits while minimizing off-target effects, even in the context of smaller vector systems like AAV vectors.
Implementation Method 1
The compact DRD is optionally an FKBP13 or a variant thereof that is responsive to a ligand such as FK506 (tacrolimus) and/or rapamycin (sirolimus). In the presence of the ligand, the payload abundance or activity increases.
Data Source
AI summary
Provided herein are engineered, regulatable polypeptides comprising a payload and a drug responsive domains (DRD), wherein the DRD is operably linked to the payload and wherein the DRD is responsive to a ligand. The DRD is optionally FKBP13, either wildtype or a variant thereof.