CD19 Primer Detection of Alternative Splice Isoforms
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Solution Overview
Problem
Current CAR-T cell therapy for B-cell malignancies faces challenges due to the down-regulation or loss of CD19 molecule expression on tumor cells, particularly through alternative spliceosomes, making it difficult to establish a stable detection system for alternatively spliced isoforms, which hinders the understanding of disease recurrence and therapy efficacy.
Innovation Solution
A method using a specific primer combination to detect CD19 expression and alternatively spliced isoforms, including primer pairs designed for exons of the CD19 gene and alternative spliceosomes, along with a reference gene like HPRT1, to quantify and amplify these isoforms accurately, facilitating the monitoring of CD19 expression and splicing mechanisms in tumor patients.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If alternative spliceosomes of CD19 mRNA are used to evade CAR-T recognition, then tumor cells can escape immune detection, but establishing a stable detection system for alternatively spliced isoforms becomes difficult
Solution Approach 1:
The patent segments the detection approach by designing specific primer pairs (EX34, EX45, J13) that target different exon regions and splice junctions of CD19 alternatively spliced isoforms. This segmentation allows the detection system to specifically identify different splicing variants (Δex2, Δex5-6, etc.) separately, enabling reliable detection despite the complexity of multiple isoforms
Solution Approach 2:
The patent applies local quality by designing primers with specific binding sites at critical regions: exon-exon junctions (J13 primer at junction of exon 1 and 3), specific exon regions (EX34 at exon 3-4, EX45 at exon 4-5). This localized primer design ensures high specificity for detecting particular alternatively spliced isoforms while maintaining system stability
2Loss of information
If alternative splicing mechanism is explored to understand disease recurrence, then understanding of therapy failure mechanisms improves, but detection and monitoring of alternatively spliced isoforms remains challenging
Solution Approach 1:
The patent implements feedback by using the detection results of alternatively spliced isoforms to guide further clinical decisions and research. The quantification of different isoform proportions provides feedback on the splicing status in tumor cells, helping to understand disease recurrence mechanisms and potentially guiding therapy adjustments
Solution Approach 2:
The patent uses PCR amplification products as an intermediary to detect alternatively spliced isoforms. The primers amplify specific regions that differ between full-length and alternatively spliced CD19 mRNA, creating detectable intermediaries (amplification products of different sizes) that reveal the splicing status without directly observing the mRNA molecules
Data Source
AI summary
The present invention belongs to the technical field of molecular biology, and specifically relates to the technical field of efficacy monitoring of a chimeric antigen receptor (CAR)-T cell therapy. The present invention provides use of a primer combination in the preparation of a product for detecting a prognostic effect of an anti-CD19 CAR-T cell therapy in a tumor, where the primer combination includes primers shown in Table 1. The present invention provides use of a primer combination in the preparation of a product for detecting an expression state of CD19 on a surface of a tumor cell in a tumor patient, where the primer combination includes primers shown in Table 1. The present invention provides a product for detecting a prognostic effect of an anti-CD19 CAR-T cell therapy in a tumor, where the product includes primers shown in Table 1 as effective components.