Retroviral Vector Cis Element Positioning
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Solution Overview
Problem
Lentiviral vector cis elements, such as the RNA packaging signal and Rev Response Element, persist in target cells after transduction, leading to issues like aberrant splicing, transgene silencing, and potential remobilization of lentiviral genomes, which complicates gene therapy applications.
Innovation Solution
The cis elements are located downstream of the 3' LTR in the retroviral vector, ensuring they are present in the viral RNA genome for packaging but excluded from the reverse-transcribed DNA in target cells, thereby minimizing their persistence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cis elements are included in the retroviral vector for packaging, then viral RNA genome packaging is enabled, but cis elements persist in target cells causing aberrant splicing and transgene silencing
Solution Approach 1:
The patent extracts the harmful cis elements (packaging signal and RRE) from the region that is reverse transcribed into DNA. By positioning these elements downstream of the 3' LTR, they are excluded from the reverse transcription process while remaining in the RNA genome for packaging. This separates the function of RNA packaging from DNA persistence, eliminating the harmful effects in target cells.
Solution Approach 2:
The patent segments the viral genome into distinct functional regions: the region upstream of the 3' LTR that is reverse transcribed into DNA, and the region downstream of the 3' LTR containing cis elements that remains in RNA form. This segmentation allows different parts of the genome to have different fates during reverse transcription, enabling packaging without persistence.
2Stability of the object's composition
If cis elements are reverse transcribed into DNA, then they are present in the provirus, but they cause remobilization of lentiviral genomes and complicate gene therapy applications
Solution Approach 1:
The patent removes cis elements from the DNA provirus by positioning them downstream of the 3' LTR, outside the reverse transcription boundary. This extraction ensures that the provirus integrates into the target cell genome without containing packaging signals or RRE, eliminating remobilization potential while maintaining essential proviral functions.
3Reliability
If cis elements are positioned between LTRs for traditional packaging, then RNA packaging is achieved, but the region size is reduced limiting transgene capacity
Solution Approach 1:
The patent relocates cis elements from the traditional position between LTRs to a new position downstream of the 3' LTR. This spatial repositioning in the genome architecture allows the cis elements to be physically separated from the reverse transcription region, effectively increasing the available space for transgene insertion upstream of the 3' LTR without compromising packaging function.
Data Source
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AI summary
There is disclosed a retroviral vector comprising a primer binding site, a long terminal repeat and an RNA packaging sequence, wherein the RNA packaging sequence is located 3' of the long terminal repeat and no long terminal repeat is located 3' of the RNA packaging sequence such that reverse transcription initiated at the primer binding site does not lead to reverse transcription of the RNA packaging sequence into vector DNA in a target cell. Also described is a host cell, a virion, a pharmaceutical composition, a method and uses including or involving the vector described above. Further, a cell or transgenic animal produced by using the vector is also described.