CRISPR-Cas9 CBLB Knockout in T Cells for Durable Immunotherapy
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Solution Overview
Problem
Current adoptive T cell therapies for cancer, including solid tumors, face challenges in achieving stable and long-term inhibition of CBLB expression, which negatively regulates immune cell costimulatory signals, leading to impaired T cell function and therapeutic efficacy.
Innovation Solution
Utilizing a CRISPR/Cas9 system for targeted genome editing in T cells to induce double-strand breaks in the CBLB gene, followed by imperfect repair through NHEJ, resulting in insertions or deletions that alter CBLB expression, thereby achieving stable repression of CBLB function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR/Cas9 system is used for targeted genome editing of CBLB gene, then CBLB expression is stably repressed and T cell function is enhanced, but the complexity of the therapy process increases
Solution Approach 1:
The CRISPR/Cas9 system is divided into separate components: guide RNA molecules targeting specific CBLB sequences and Cas9 nuclease, which can be delivered independently or as pre-formed complexes. This segmentation allows for controlled action and reduces off-target effects while maintaining stable CBLB repression.
Solution Approach 2:
Ribonucleoprotein (RNP) complexes of Cas9 and guide RNA are pre-assembled before delivery to T cells. This preliminary formation ensures precise targeting and immediate upon delivery, reducing the time the editing machinery remains active in the cell and minimizing potential side effects while achieving stable CBLB knockout.
2Productivity
If double-strand breaks are induced in CBLB gene to achieve stable repression, then T cell proliferation and survival are improved, but the risk of off-target effects and genomic instability increases
Solution Approach 1:
Guide RNA molecules serve as intermediaries that direct the Cas9 nuclease to specific CBLB gene sequences. The guide RNA contains sequence complementary to CBLB target sites, ensuring precise localization of the nuclease activity and minimizing off-target DNA breaks through specific base pairing requirements.
Solution Approach 2:
The CRISPR/Cas9 editing components, particularly the RNP complexes, are designed to be transient rather than permanent. The complexes perform their editing function and then degrade, leaving no persistent foreign DNA in the T cells. This disposable approach reduces long-term genomic instability risks while achieving the desired CBLB knockout effect.
3Reliability
If CBLB expression is inhibited to enhance T cell function, then therapeutic efficacy against cancer is improved, but T cell dependency on co-stimulation signals increases
Solution Approach 1:
The patent converts the harmful negative regulatory function of CBLB into a benefit by selectively knocking out CBLB in T cells. This knockout removes the brake on T cell activation, allowing enhanced responses to tumor antigens. The therapy exploits this converted benefit to improve anti-tumor efficacy while managing co-stimulation requirements through controlled delivery and timing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The CRISPR/Cas9 system enables long-term reduction of CBLB expression, enhancing T cell proliferation, survival, and function, particularly in the absence of CD28 co-stimulation and cytokines, improving therapeutic outcomes in cancer treatment.
Implementation Method 1
the alteration in CBLB expression occurs as a result of a double-stranded break induced by the RNP and subsequent imperfect repair that leads to indels at and/or adjacent to the targeted CBLB sequence
Implementation Method 2
a guide RNA with a targeting domain that is complementary to target sequence of a CBLB gene
Data Source
AI summary
CRISPR/CAS-related genome editing systems, compositions and methods for targeting the CBLB locus, as well as cells edited using these systems, compositions and methods are provided.