Multicistronic Expression Constructs with Hairpin RNA
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Solution Overview
Problem
Multicistronic expression constructs often face reduced expression levels due to promoter interference and the presence of incompatible nucleic acid elements, particularly when including a hairpin RNA expression cassette, which can interfere with viral vector reproduction or packaging, especially in viral vectors like AAV.
Innovation Solution
The use of isolated nucleic acid constructs with a first expression cassette containing a gene product under a promoter and a second self-complementary expression cassette, such as a hairpin RNA, positioned within an intron of the first cassette, either in the same or opposite orientation, and in proximity to an AAV-ITR, particularly a ΔTRS ITR, to enhance expression efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a multicistronic expression construct includes a hairpin RNA expression cassette, then the delivery of non-translated gene products together with a reporter transgene is enabled, but the expression levels of the included transgenes are reduced due to promoter interference or the presence of incompatible nucleic acid elements in close proximity
Solution Approach 1:
The patent divides the multicistronic expression construct into separate expression cassettes, each with its own promoter and coding sequence. This segmentation allows independent optimization of each cassette to avoid promoter interference while maintaining the ability to deliver multiple transgenes simultaneously from a single viral vector.
Solution Approach 2:
The patent introduces self-complementary nucleic acid sequences as intermediary elements between expression cassettes. These sequences act as spacers that reduce interference between adjacent cassettes while maintaining proper spacing for viral packaging, thereby preserving expression levels without compromising delivery capability.
2Adaptability or versatility
If a multicistronic expression construct is part of a viral vector, then the delivery of multiple transgenes is enabled, but the inclusion of a hairpin RNA expression cassette may interfere with the formation of structures necessary for viral reproduction or packaging
Solution Approach 1:
The patent applies local quality by positioning self-complementary nucleic acid sequences specifically at critical locations within the viral vector construct - namely, in proximity to the inverted terminal repeats (ITRs). This localized placement ensures that the hairpin RNA expression cassette does not interfere with viral packaging structures while still enabling multi-transgene delivery.
Solution Approach 2:
The patent modifies the physical parameters of the nucleic acid construct by controlling the distance and orientation of self-complementary sequences relative to the ITRs. By adjusting these spatial parameters, the construct maintains compatibility with viral packaging mechanisms while enabling the inclusion of hairpin RNA cassettes for enhanced delivery versatility.
Data Source
AI summary
Some aspects of this invention provide nucleic acid constructs for transgene expression. Some aspects of this invention provide multicistronic nucleic acid constructs, for example, comprising an expression cassette encoding a hairpin RNA and a reporter expression cassette. Some aspects of this invention provide nucleic acid constructs comprising two or more self-complementary nucleic acid sequences, for example, hairpin RNA encoding nucleic acid sequences and AAV inverse terminal repeats. Methods for the use of the constructs in therapy and research are also provided.