Viral Vector ITR-Adjacent Termination for Off-Target Expression Control
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Solution Overview
Problem
Existing gene therapy and genetic engineering methods face challenges in controlling undesired or off-target gene expression, leading to potential toxicity and reduced effectiveness due to inappropriate gene expression.
Innovation Solution
Recombinant viral vectors are designed with transcription termination sequences adjacent to inverted terminal repeat sequences, flanking expression cassettes, to control and reduce off-target gene expression, enhancing specificity and dosage efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If transcription termination sequences are added adjacent to ITR sequences to reduce off-target gene expression, then specificity of gene expression is improved, but device complexity increases
Solution Approach 1:
The patent extracts the transcription termination function from the expression cassette and places it adjacent to the ITR sequences. This separates the termination function from the gene expression control elements, allowing it to operate independently to prevent read-through transcription into adjacent sequences, thereby reducing off-target expression without interfering with the intended gene expression control
Solution Approach 2:
The transcription termination sequence acts as an intermediary element between the ITR sequences and the expression cassette. It mediates the transcription process by providing a termination signal that stops RNA polymerase before it can transcribe into adjacent sequences, thus preventing unwanted off-target gene expression while maintaining the integrity of the intended expression cassette
2Object-generated harmful factors
If transcription termination sequences are placed adjacent to ITR sequences, then off-target gene expression is reduced, but loss of information increases due to potential interference with regulatory elements
Solution Approach 1:
The patent applies local quality by placing the transcription termination sequence specifically at locations adjacent to ITR sequences where it is needed to prevent read-through transcription, while leaving other regions of the vector unchanged. This localized approach ensures that termination occurs only where necessary to prevent off-target expression, without interfering with the function of regulatory elements within the expression cassette
Solution Approach 2:
The transcription termination sequence is positioned upstream of potential off-target transcription regions, performing the termination action before unwanted transcription can occur. This preliminary placement of the termination signal prevents read-through transcription into adjacent sequences or regulatory elements, ensuring that the intended gene expression is not affected while blocking off-target expression
Data Source
AI summary
The present disclosure provides viral vectors which decrease undesired or off-target gene expression. Particularly, the present disclosure provides recombinant viral vectors comprising a transcription termination sequence adjacent to an inverted terminal repeat sequence (e.g., downstream of a first inverted terminal repeat sequence and upstream of an expression cassette), and virus or virus-like particles, compositions, and methods of using thereof.