Antisense Transfer Vector for T Cell Gene Co-Expression
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Solution Overview
Problem
Current methods for co-expression of genes like CAR or TCR and gene cargo in T cells face challenges due to high costs and risks of insertional mutagenesis, and existing strategies like IRES and P2A sequences have drawbacks such as large size, cell-type dependency, and immunogenicity.
Innovation Solution
A polynucleotide comprising two gene cassettes in antisense orientation, with one cassette encoding a CAR or TCR and the other encoding a gene cargo, linked to constitutive or inducible promoters, allowing for independent expression and reduced immunogenicity, and arranged between LTRs for efficient lentiviral vector production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If IRES or P2A sequences are used for co-expression of genes, then gene co-expression is achieved, but device complexity and immunogenicity increase
Solution Approach 1:
The patent divides the polynucleotide into two separate sense-oriented gene cassettes (first gene cassette and second gene cassette) that are transcribed independently. Each cassette contains its own promoter, coding sequence, and polyadenylation signal, eliminating the need for complex IRES or P2A sequences while achieving independent co-expression of multiple genes.
2Ease of manufacture
If sense orientation is used for both gene cassettes, then expression is simplified, but transcriptional interference occurs reducing expression levels
Solution Approach 1:
The patent employs asymmetric orientation where the first gene cassette is in sense orientation and the second gene cassette is in antisense orientation relative to each other. This asymmetric arrangement prevents transcriptional interference between the two cassettes while maintaining ease of construction, as each cassette retains its own promoter and terminates with its own polyadenylation signal.
Data Source
AI summary
The present disclosure describes a next-generation antisense transfer vector along with methods for a high titer lentivirus production, allowing efficient transduction of T cells with a constitutively expressed tumor-targeting receptor along with the activation-induced expression of various gene cargos. The disclosed antisense transfer vector and the methods can reduce virus production costs as well as enhance the efficacy and safety of next-generation CAR- or TCR-T cells reaching the clinic.