Polycistronic Vectors with 2A Peptides for Homogeneous CAR-T Expression
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Solution Overview
Problem
Current methods for co-expressing multiple genes in cells using multiple vectors result in heterogeneous cell populations, leading to diminished persistence of engineered cell phenotypes, complex manufacturing, and lot-to-lot variability, which are unsuitable for therapeutic applications.
Innovation Solution
Development of polycistronic vectors that include a polycistronic expression cassette with a chimeric antigen receptor (CAR) for CD19, a fusion protein of IL-15 and IL-15Rα, and a marker protein, separated by F2A and T2A elements, to ensure uniform expression across a cell population.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple vectors are used to co-express multiple genes in single cells, then gene co-expression capability is achieved, but cell population homogeneity deteriorates
Solution Approach 1:
The patent merges multiple gene expression cassettes into a single polycistronic vector. The self-cleaving peptide elements (2A peptides) enable the single vector to produce multiple separate proteins from one transcript, achieving gene co-expression while maintaining cell population homogeneity by ensuring all cells receive the same genetic material
2Adaptability or versatility
If multiple vectors are used for transgene delivery, then multiple genes can be introduced, but manufacturing complexity increases
Solution Approach 1:
The patent combines multiple transgenes into a single polycistronic expression cassette within one vector. The 2A peptide-mediated cleavage allows one vector to deliver multiple genes that are expressed as separate proteins, simplifying manufacturing by eliminating the need to produce, purify, and transfect multiple separate vectors
3Adaptability or versatility
If multiple vectors are employed for gene co-expression, then transgene diversity is achieved, but product consistency deteriorates
Solution Approach 1:
The patent merges multiple transgenes into a single polycistronic construct where each gene is separated by 2A peptide elements. This ensures that all cells receive identical genetic material and express all transgenes at comparable levels, achieving product consistency while maintaining transgene diversity for complex therapeutic functions
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 polycistronic vectors achieve homogeneous expression of multiple proteins in immune effector cells, enhancing therapeutic efficacy and simplifying manufacturing processes by ensuring consistent expression levels across the cell population.
Implementation Method 1
the polynucleotide encoding the anti-CD19 CAR is separated from the polynucleotide encoding the fusion protein by a polynucleotide sequence that comprises an F2A element, and the polynucleotide encoding the fusion protein is separated from the polynucleotide sequence encoding the marker protein by a polynucleotide sequence that comprises a T2A element
Data Source
AI summary
Provided herein are vectors comprising a polycistronic expression casstte comprising a polynucleotide that encodes a CD 19 specific chimeric antigen receptor, a polynucleotide that encodes a cytokine, and a polynucleotide that encodes a marker protein, wherein the polynucleotide that encodes the CD 19-specific chimeric antigen receptor and the polynucleotide that encodes the cytokine coding sequence are separated by a polynucleotide sequence that comprises an F2A element, and wherein the polynucleotide sequence that encodes the cytokine and the polynucleotide sequence that encodes the marker protein are separated by a polynucleotide sequence that comprises a T2A element.