In Situ Gene Editing via AAV Delivery to Native Stem Cells
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Solution Overview
Problem
Current gene editing methods, particularly CRISPR/cas9 based, require ex vivo manipulation of cells, which is limited by the inability to modify solid tissues in vivo and poses risks such as graft failure and disruption of native stem cell niches.
Innovation Solution
An AAV-based in vivo system for programmable DNA modifying enzymes that allows direct transduction of endogenous tissue stem cells, preserving native regulatory interactions and avoiding the need for cell isolation or transplantation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If ex vivo cell isolation and transplantation methods are used for gene editing, then cell modification can be achieved, but native stem cell niches are disrupted and graft failure risk increases
Solution Approach 1:
The patent uses viral vectors (AAV, lentivirus, retrovirus) as intermediaries to deliver gene editing tools directly to stem cells in their native tissue environment. These vectors serve as mediators that can cross the blood-tissue barrier and deliver payloads without requiring cell isolation, thereby preserving native regulatory interactions while achieving gene modification.
Solution Approach 2:
The patent replaces the mechanical process of cell isolation, culture, and transplantation with a biochemical approach using viral vectors for in vivo delivery. This substitution eliminates the need to physically remove cells from their niches, maintaining the structural and functional integrity of stem cell ecosystems while achieving genetic modification.
2Ease of manufacture
If solid tissues are removed from patients for ex vivo modification, then gene editing can be performed, but tissue function is disrupted and graft failure occurs
Solution Approach 1:
Viral vectors serve as intermediaries that can deliver gene editing tools through the blood-tissue barrier into solid tissues without requiring surgical removal. This allows gene editing to be performed in situ, maintaining tissue integrity and function while avoiding the risks of graft failure associated with tissue removal and reimplantation.
3Adaptability or versatility
If multiple distinct genetically engineered alleles are generated for transgenic approaches, then gene disruption can be achieved, but breeding complexity and time requirements increase
Solution Approach 1:
The patent performs gene editing in advance within living organisms using in vivo viral vector delivery, eliminating the need for complex breeding schemes to generate multiple alleles. The gene editing tools are delivered directly to target cells before the desired genetic modification is needed, saving significant time that would otherwise be required for breeding multiple distinct genetically engineered mice.
Solution Approach 2:
The patent replaces the mechanical and time-consuming process of generating and breeding multiple distinct alleles with a direct in vivo gene editing approach using viral vectors. This biochemical method achieves the same gene disruption capability without requiring the complex breeding procedures that would otherwise be necessary to create and maintain multiple transgenic lines.
4Ease of manufacture
If ex vivo stem cell modification is performed, then gene editing can be achieved, but expensive GMP facilities are required and cell handling risks increase
Solution Approach 1:
Viral vectors serve as intermediaries that enable gene editing to occur in vivo, eliminating the need for expensive GMP facilities and specialized cell handling infrastructure. The vectors can be administered through standard routes (intravenous, intraperitoneal, local injection), allowing gene editing to be performed in conventional laboratory settings rather than requiring high-containment facilities.
Solution Approach 2:
The patent replaces the complex mechanical system of ex vivo cell culture, purification, and transplantation with a simpler in vivo delivery approach using viral vectors. This substitution eliminates the need for GMP facilities and specialized cell handling equipment, reducing infrastructure requirements while maintaining gene editing capability.
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
Enables efficient and safe genome modification of various cell types in situ, overcoming technological limitations of ex vivo methods and allowing rapid interrogation of gene functions and interactions across different animal ages and strains.
Implementation Method 1
contacting the subject with a virus, wherein the virus transduces a nucleic acid sequence encoding a sequence-targeting nuclease into the population of cells
Data Source
AI summary
Disclosed are methods of in situ genomic modification of cells (e.g., stem cells, tissue stem cells, muscle stem cells, Sca-1+ mesenchymal progenitor cells in skeletal muscle, CD140a+ dermal mesenchymal cells) using sequence-targeting nucleases delivered via a virus (e.g., an AAV).