Compact Bidirectional Promoters for AAV Gene Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene editing systems, such as CRISPR/Cas9, face challenges in delivering both nuclease and guide RNA components within the size limits of AAV vectors, limiting their application to specific cell and tissue types due to space constraints.
Innovation Solution
Development of compact, bidirectional promoters that allow for the expression of both nuclease and guide RNA in a single vector, such as an AAV vector, by utilizing regulatory elements that direct transcription in opposite directions, enabling efficient packaging and expression in various cell types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional promoters are used to express Cas9 and gRNA, then both components can be expressed, but the total DNA size exceeds the AAV packaging limit of 4.7-4.9 kb
Solution Approach 1:
The patent applies parameter changes by replacing conventional large promoters (500 bp each) with compact promoters (100-200 bp) that maintain bidirectional transcription capability. This size reduction of approximately 60-80% in promoter length allows the total construct to fit within the AAV packaging limit while preserving the ability to express both Cas9 and gRNA components in various cell and tissue types.
2Quantity of substance
If a two-vector approach is used to deliver Cas9 and gRNA, then packaging capacity is sufficient, but the system requires two separate vectors which complicates delivery
Solution Approach 1:
The patent merges the delivery of Cas9 and gRNA into a single AAV vector by using compact bidirectional promoters. This combining of two separate expression cassettes into one vector simplifies the delivery system, eliminating the need for dual-vector co-delivery while maintaining the ability to express both components. The compact promoter design enables both genes to be packaged together within the 4.7-4.9 kb capacity limit.
3Quantity of substance
If the murine Mecp2 promoter is used to reduce DNA size, then the promoter length is minimized, but expression is limited to specific retinal cell types and not rods
Solution Approach 1:
The patent achieves universality by designing compact promoters that maintain broad tissue and cell type expression capability while minimizing size. Unlike the murine Mecp2 promoter which is restricted to specific retinal regions, the compact promoters described (100-200 bp) are engineered to drive expression across diverse cell and tissue types including rods, thereby providing both size efficiency and broad applicability.
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 the inclusion of both nuclease and guide RNA in a single vector, expanding the applicability of gene editing systems to a broader range of cell and tissue types beyond previous limitations.
Implementation Method 1
a) at least one regulatory element that provides for transcription in one direction of at least one nucleotide sequence encoding a guide RNA (gRNA)... and b) at least one regulatory element that provides for transcription in the opposite direction of a nucleotide sequence encoding a nuclease
Data Source
AI summary
The invention relates generally to compact promoters and their use in gene editing e.g., for treating disease. The disclosure is based, in part, upon the discovery of compact, bidirectional promoters that can be used to express both a nuclease (e.g., a Cas9 nuclease) and a guide RNA (gRNA). For example, in certain embodiments disclosed herein, a compact, bidirectional promoter can comprise at least one regulatory element that directs expression of a gRNA in one direction and at least one regulatory element that directs expression of a nuclease in the other direction. Accordingly, the promoters disclosed herein use less space than prior art promoters, allowing both a nuclease and a gRNA to be packaged in a single vector (e.g., a plasmid or an AAV).