Fas Splicing Modulation in T Cells for Exon 6 Skipping
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing immunotherapies, such as adoptive-cell therapy and CAR-T cells, face limitations in effectively targeting and eliminating cancer cells due to the dominance of membrane-bound Fas (mFas) over the soluble Fas (sFas) form, which can lead to suboptimal immune cell functionality and sustainability.
Innovation Solution
Manipulating T cells to enhance skipping of Fas exon 6, promoting the expression of sFas through the use of splicing modulating agents like splice-switching oligonucleotides (SSOs) and CRISPR/Cas9 systems, thereby altering Fas splicing to predominantly express sFas.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If membrane-bound Fas (mFas) is dominant in T cells, then Fas-mediated death signaling is strong, but immune cell functionality and sustainability are reduced
Solution Approach 1:
The patent changes the expression parameter of Fas isoforms by manipulating splicing to shift the balance from membrane-bound Fas (mFas) to soluble Fas (sFas). This parameter change in Fas isoform expression resolves the contradiction by reducing mFas dominance while enhancing immune cell functionality and sustainability through increased sFas production.
Solution Approach 2:
Instead of increasing mFas expression to enhance death signaling, the patent inverts the approach by decreasing mFas and increasing sFas. This inversion resolves the contradiction by using sFas (the alternative form) to achieve the desired immune cell functionality without the harmful effects of mFas dominance.
2Duration of action of stationary object
If soluble Fas (sFas) expression is increased through exon 6 skipping, then immune cell sustainability is improved, but splicing manipulation complexity increases
Solution Approach 1:
The patent uses splicing modulating agents (SMAs) as intermediary molecules to manipulate Fas splicing. These SMAs act as mediators that guide the splicing process to skip exon 6 and produce sFas, thereby improving immune cell sustainability while managing the complexity through a defined molecular mechanism.
Solution Approach 2:
The patent changes the splicing parameter of the Fas gene to favor exon 6 skipping, which produces sFas. This parameter change in the splicing process directly improves immune cell sustainability and duration of action, while the change itself provides a controllable mechanism for achieving the desired outcome.
3Productivity
If Fas splicing is manipulated to enhance sFas expression, then cytokine secretion and activation marker expression increase, but manufacturing precision requirements increase
Solution Approach 1:
The patent employs splicing modulating agents as intermediary substances that precisely control Fas splicing. These SMAs serve as mediators that ensure accurate exon 6 skipping and sFas production, thereby enhancing cytokine secretion and activation marker expression while maintaining manufacturing precision through a controlled splicing mechanism.
Solution Approach 2:
The patent implements feedback control in the splicing manipulation process to ensure accurate production of sFas. By monitoring and adjusting splicing parameters, the system maintains precision while maximizing cytokine secretion and activation marker expression, resolving the contradiction between productivity and manufacturing precision.
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
Enhances immune cell functionality and sustainability by increasing sFas expression, leading to improved cytokine secretion, activation marker expression, and increased killing of target cells, with potential benefits in adoptive cell therapy for cancer treatment.
Implementation Method 1
During the transcription process, the introns of the pre-mRNA are spliced out, and the exons are joined together, resulting in the mature mRNA. In 95% of the genes, side by side with the constitutive mRNA form, skipping over exons (or part of them) can lead to a different configuration of mRNA, significantly increasing the diversity of proteins. This process is called alternative splicing (AS).
Implementation Method 2
FAS is mainly known for its death signal transduction following FAS ligand (FASL) binding. However, it also has other functions, e.g., it takes part in the differentiation of naïve T to memory T cells.
Data Source
AI summary
The present disclosure relates to manipulation of splicing of the Fas (tumor necrosis factor (TNF) receptor superfamily, member 6) gene in non-naïve cell of the T lineage for enhanced skipping of exon 6. The manipulated non-naïve cells predominantly express a soluble form of Fas (sFas) and display reduced expression of the membranal Fas (mFas), increased cytokine secretion, increased expression of activation markers, increased cell survival, increased cytotoxicity, and/or reduced expression of exhaustion markers. The present disclosure further provides specific splicing modulatory agents comprising gene editing systems and/or splice switching antisense oligonucleotide (SSO), methods and therapeutic uses thereof.