LiBIS-seq Cell-free DNA Insertion Site Sequencing
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Solution Overview
Problem
Current methods for analyzing insertion sites in gene therapy are limited, as they primarily rely on invasive tissue biopsies and do not effectively monitor vector-marked cell clones in solid organs, leading to potential late detection of adverse effects such as malignancy.
Innovation Solution
The development of Liquid Biopsy Insertion Site Sequencing (LiBIS-seq) method, which identifies and quantifies viral and vector insertion sites in cell-free DNA from body fluids like blood plasma, allowing non-invasive monitoring of insertion sites in both circulating and solid organ cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If invasive tissue biopsies are used to analyze insertion sites, then direct detection of vector-marked cell clones in solid organs is achieved, but patient safety is compromised and the procedure becomes risky
Solution Approach 1:
The patent uses cell-free DNA from body fluids as a copy of the genomic material to indirect detection of insertion sites. Instead of directly biopsying solid organs, the method sequences insertion sites from circulating cell-free DNA, which contains molecular copies of integrated vector sequences, thereby achieving detection without tissue invasion
Solution Approach 2:
The patent introduces body fluids (blood, plasma, serum) as an intermediary medium to access vector-marked cell clones. Cell-free DNA in these fluids serves as a accessible proxy that reflects the presence of integrated vectors in solid organs without requiring direct tissue sampling
2Reliability
If invasive tissue biopsies are performed to monitor solid organ cells, then comprehensive monitoring of vector-marked clones is achieved, but the procedure becomes complex and risky
Solution Approach 1:
The method creates molecular copies of insertion sites through PCR amplification of cell-free DNA fragments containing vector integration junctions. This copying process enables repeated, non-invasive monitoring of the same insertion sites over time without requiring successive biopsies
Solution Approach 2:
The body's own circulating cell-free DNA serves as the sample source, eliminating the need for external intervention to obtain tissue samples. The method leverages naturally present cell-free DNA in body fluids for self-monitoring of gene therapy outcomes
3Ease of operation
If cell-free DNA from body fluids is used to identify insertion sites, then non-invasive monitoring is achieved, but detection sensitivity may be reduced
Solution Approach 1:
The method performs preliminary enrichment and purification of cell-free DNA from body fluids before sequencing. By concentrating and preparing the DNA sample through specific protocols, the method enhances the quality and quantity of analyzable material to improve detection sensitivity
Solution Approach 2:
The patent employs PCR amplification to create multiple copies of the target insertion site sequences from limited cell-free DNA input. This partial amplification approach concentrates the signal of interest while filtering out background noise, thereby enhancing detection sensitivity despite the small initial sample amount
Data Source
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AI summary
A method for analysing insertion sites of an exogenous nucleotide sequence in a subject's genome, wherein the method comprises: (a) providing a sample from the subject comprising cell-free double-stranded DNA polynucleotides; (b) blunting the ends of the polynucleotides; (c) ligating an oligonucleotide to both ends of the polynucleotides; (d) amplifying polynucleotides comprising an insertion site; and (e) sequencing the product of step (d).