Minimal U6 Pol III Promoter for Larger Viral Vector Payloads
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing type III RNA polymerase promoters, such as H1 and U6, are too long and limit the size and number of nucleic acid constructs that can be incorporated into viral vectors, restricting the expression of RNA sequences like siRNA, miRNA, and CRISPR guide sequences.
Innovation Solution
Development of a minimal U6 pol III promoter, reduced to 74-117 bp, which includes a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box, allowing for increased space in viral vectors for larger or multiple nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a wild-type U6 pol III promoter is used, then proper transcription function is achieved, but the promoter length (around 300 bp) limits the size and number of nucleic acid constructs that can be incorporated into viral vectors
Solution Approach 1:
The patent extracts and removes the nucleosomal region (intervening sequence of around 150 bp) from the wild-type U6 promoter while retaining the essential functional elements (DSE, PSE, and TATA box). This extraction reduces the promoter length from approximately 300 bp to a minimal functional length, thereby resolving the contradiction between maintaining transcription function and reducing promoter size to accommodate larger nucleic acid constructs in viral vectors.
2Length of stationary object
If the promoter length is reduced to increase vector capacity, then more space is available for nucleic acid sequences, but the promoter may lose proper transcription function
Solution Approach 1:
The patent systematically removes only the non-essential nucleosomal region while preserving all critical transcriptional control elements including the distal sequence element (DSE) with its conserved octamer sequence, the proximal sequence element (PSE), and the TATA box. This selective extraction ensures that the minimal promoter maintains proper transcription function while achieving the desired length reduction.
Solution Approach 2:
The patent changes the length parameter of the promoter by deleting the intervening nucleosomal sequence, transforming it from a wild-type promoter of around 300 bp to a minimal promoter of approximately 150-200 bp. This parameter change is achieved through precise deletion of non-essential regions while maintaining the functional integrity of the remaining elements through their optimized arrangement and conservation of critical sequences.
3Length of stationary object
If a shortened H1 promoter is used, then promoter length is reduced, but the natural human U6 promoter requires an intervening sequence of around 150 bp to achieve proper co-localization of octamer element and PSE
Solution Approach 1:
The patent extracts and removes the problematic nucleosomal region that caused the need for a 150 bp intervening sequence in natural U6 promoters. By eliminating this specific structural element, the patent simplifies the promoter architecture while maintaining functionality, thereby resolving the contradiction between length reduction and structural complexity.
Solution Approach 2:
The patent segments the promoter into its essential functional components (DSE, PSE, TATA box) and removes the non-essential nucleosomal region. This segmentation allows the promoter to be shortened without losing function, as each remaining segment performs its specific role in transcriptional control independently of the removed nucleosomal structure.
Data Source
AI summary
The invention relates to a minimal U6 pol III promoter. The invention also concerns a nucleic acid construct comprising the minimal U6 pol III promoter, a vector comprising the minimal U6 pol III promoter, methods involving the minimal U6 pol III promoter, and uses for the minimal U6 pol III promoter.