Activation Inducible Immune Cell Expression System
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Solution Overview
Problem
Current methods for genetically modifying immune cells, such as T cells, for adoptive cell therapies face challenges including unpredictable transgene expression due to random integration, potential oncogenic risks, and the inefficiencies and costs associated with viral vector-based delivery methods, as well as the need for tighter control over transgene expression in an activation-dependent manner.
Innovation Solution
The method involves site-specific insertion of transgenes at targeted loci within the immune cell genome, using CRISPR-Cas9 mediated gene editing, with non-viral polynucleotides and electroporation, allowing for controlled expression under endogenous promoters like IL-13 or GM-CSF, reducing random integration and enabling activation-dependent transgene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If viral vectors are used to deliver transgenes to immune cells, then transgene delivery is achieved, but the process becomes time-consuming (>6 months), expensive and poses biosafety challenges
Solution Approach 1:
The patent extracts and eliminates the viral vector component from the transgene delivery system. Instead of using viral vectors (retrovirus, lentivirus) that require complex production processes taking >6 months, the invention employs direct non-viral DNA delivery methods with electroporation, reducing production time to weeks while maintaining transgene delivery effectiveness
Solution Approach 2:
The patent replaces expensive, complex viral vector production systems with simpler, more economical non-viral DNA delivery approaches. The plasmid DNA templates can be produced quickly and cost-effectively, eliminating the need for expensive viral vector manufacturing infrastructure and reducing overall production costs
2Reliability
If viral vectors are used to deliver transgenes to immune cells, then transgene delivery is achieved, but the process becomes expensive and poses biosafety challenges
Solution Approach 1:
The patent removes the viral vector system entirely, replacing it with direct non-viral DNA delivery. This extraction eliminates the expensive viral vector production infrastructure, licensing costs, and biosafety compliance requirements while maintaining effective transgene delivery through electroporation-based methods
Solution Approach 2:
The invention uses inexpensive plasmid DNA templates that can be produced through standard bacterial transformation and purification protocols, replacing expensive viral vector production. The non-viral DNA delivery system reduces costs related to viral vector manufacturing, quality control, and biosafety compliance
3Reliability
If random integration of transgenes is used, then transgene expression is achieved, but expression becomes unpredictable and variable
Solution Approach 1:
The patent applies local quality by targeting specific genomic loci for transgene integration rather than allowing random integration. The transgene is inserted at predetermined locations such as the IL-13 locus or other suitable genomic sites, ensuring consistent expression patterns and avoiding the variability associated with random integration positions
Solution Approach 2:
The patent performs preliminary action by pre-identifying and preparing specific target loci for transgene integration. The genomic architecture at these predetermined sites is optimized in advance to ensure consistent transgene expression, eliminating the need to wait for random integration events and their subsequent variability
4Reliability
If random integration of transgenes is used, then transgene expression is achieved, but malignant transformation risk increases if integrated into oncogenic loci
Solution Approach 1:
The patent applies local quality by selecting specific, safe genomic loci for transgene integration that are known not to disrupt oncogenic pathways. Target sites such as the IL-13 locus are chosen for their benign genomic context, eliminating the risk of inserting transgenes into oncogenic loci that could cause malignant transformation
Solution Approach 2:
The patent converts the potential harm of random integration (which could land in oncogenic loci) into a benefit by using targeted integration approaches. The same gene editing machinery (CRISPR-Cas9) that could cause harmful random integration is directed toward specific safe loci, transforming a risky process into a safe and controlled one
5Manufacturing precision
If site-specific integration is used, then integration precision is improved, but the method requires optimization for better knock-in efficiencies and reduced toxicity
Solution Approach 1:
The patent applies parameter changes by optimizing multiple variables in the site-specific integration process: adjusting CRISPR-Cas9 component concentrations, modifying electroporation parameters (voltage, pulse duration), adjusting DNA template concentrations and sequences, and controlling cell cycle conditions. These parameter optimizations achieve high knock-in efficiencies at target loci while maintaining safety
6Reliability
If viral vectors are used for donor DNA template delivery, then gene editing is achieved, but the process becomes time consuming, expensive and labor-intensive
Solution Approach 1:
The patent extracts and removes the viral vector delivery component from the gene editing process. Instead of using complex viral vector systems for delivering donor DNA templates, the invention employs direct non-viral DNA delivery through electroporation, significantly simplifying the overall process while maintaining gene editing effectiveness
Solution Approach 2:
The patent replaces complex, expensive viral vector production systems with simple, inexpensive plasmid DNA templates. The non-viral DNA delivery approach eliminates the need for viral vector manufacturing infrastructure, reducing both complexity and cost while achieving the same gene editing outcomes
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
This approach enhances the efficiency and safety of transgene integration, reduces toxicity, and allows for precise control of transgene expression, improving the efficacy of immune cell therapies by ensuring targeted and controlled activation-dependent function.
Implementation Method 1
Site-specific gene integration can be achieved with the use of gene-editing tools (e.g., Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, Transcription activator-like effector nuclease (TALEN), Zinc finger nuclease (ZFN), Meganucleases), which results in DNA double-strand breaks (DSBs) and homology-directed repair (HDR) when a donor DNA template is provided.
Implementation Method 2
Various delivery approaches have been employed to deliver the donor DNA template [13-17], including the use of viral vectors. In one embodiment, the donor polynucleotide is delivered to the immune cell using electroporation.
Data Source
AI summary
The present invention provides methods of genetically modifying an immune cell such that the immune cell expresses a transgene in an activation dependent manner. The application also provides genetically modified immune cells prepared using such methods, and the uses of the genetically modified immune cells in immunotherapy (e.g., adoptive cell therapy) for treatment of a disease such as cancer, autoimmune disease or infectious disease.