Modified AAV Constructs Using Pri-miRNA Scaffolds for Gene Silencing
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Solution Overview
Problem
Existing AAV-delivered short-hairpin RNAs (shRNAs) cause cellular toxicity due to perturbation of the RNA interference machinery, and reducing shRNA levels or using less efficient serotypes or weaker promoters negatively impact RNAi potency.
Innovation Solution
Engineering recombinant Adeno-associated viruses (rAAVs) to express inhibitory RNA guide strands within artificial miRNA scaffolds, such as mouse pri-miRNA-33, to target human genes like SOD1 or PC-1, while using promoters like U6 or CBA to maintain effective gene knockdown with reduced off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high levels of AAV-delivered shRNAs are used, then gene knockdown potency is improved, but cellular toxicity increases due to perturbation of the RNA interference machinery
Solution Approach 1:
The patent changes the structural parameters of the shRNA molecule by embedding it within a pri-miRNA scaffold structure. This structural transformation allows the shRNA to be processed through the natural miRNA pathway, reducing perturbation of the RNAi machinery while maintaining gene silencing potency. The scaffold structure modifies how the shRNA is recognized and processed by cellular enzymes, thereby reducing toxicity.
Solution Approach 2:
The pri-miRNA scaffold acts as an intermediary structure that mediates between the shRNA sequence and the cellular processing machinery. Instead of directly introducing shRNA that perturbs the RNAi pathway, the scaffold provides a natural-looking structure that is processed by Drosha and Dicer enzymes, thereby reducing cellular stress while still delivering the gene-silencing function.
2Object-affected harmful factors
If vector doses are reduced to lower shRNA levels, then cellular toxicity is reduced, but RNAi potency is negatively impacted
Solution Approach 1:
The patent changes the efficiency parameter of shRNA expression by using a pri-miRNA scaffold that is processed more efficiently by cellular machinery. The scaffold structure enables better incorporation into the RNA-induced silencing complex (RISC), thereby achieving effective gene knockdown at lower expression levels and reducing the need for high vector doses.
3Object-affected harmful factors
If weaker Pol II promoters are used instead of strong Pol III promoters, then cellular toxicity is reduced, but RNAi potency is negatively impacted
Solution Approach 1:
The patent changes the promoter compatibility by designing the shRNA expression cassette to function effectively with Pol II promoters through the pri-miRNA scaffold structure. The scaffold contains elements that are recognized by Pol II transcription machinery, allowing efficient transcription and processing while enabling the use of weaker, more controllable promoters that reduce cellular toxicity.
4Object-affected harmful factors
If less efficient AAV serotypes are used, then cellular toxicity is reduced, but RNAi potency is negatively impacted
Solution Approach 1:
The patent changes the efficiency of the RNAi mechanism itself through the pri-miRNA scaffold structure, rather than relying on serotype efficiency. The scaffold improves processing efficiency and RISC loading, thereby achieving potent gene knockdown with various AAV serotypes without needing to select for highly efficient but potentially more toxic serotypes.
Data Source
AI summary
In some aspects the disclosure relates to recombinant adeno-associated virus (rAAV) vectors and rAAVs (e.g., viral particles) engineered to express a transgene comprising an inhibitory nucleic acid (e.g., an artificial miRNA, amiRNA) having a pri-miRNA scaffold and a guide strand that targets a human target gene.