Toxic Gene Viral Vectors Intron Splicing Control
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Solution Overview
Problem
Current methods for producing viral vectors harboring toxic genes, such as diphtheria toxin A (DT-A), in insect cells are hindered by the toxicity of these genes, leading to nonspecific cell killing and low yields, limiting their application in cancer therapy.
Innovation Solution
Incorporating an intron into the toxic gene sequence that is spliced in mammalian cells but not in insect cells, allowing for the production of viral vectors like baculovirus and adeno-associated virus (AAV) in insect cells without toxicity, and subsequent expression of toxic proteins in mammalian cells to induce cell death.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If toxic genes are introduced into viral vectors for cancer therapy, then therapeutic efficacy is improved, but host cell viability deteriorates leading to low vector production yields
Solution Approach 1:
The patent divides the toxic gene expression function into two separate segments: (1) the viral vector production phase in insect cells where the toxic gene is inactive, and (2) the therapeutic action phase in mammalian cells where the toxic gene becomes active. This segmentation allows high-yield vector production without toxicity, followed by specific therapeutic action in target cells.
Solution Approach 2:
The patent uses an intron as an intermediary element that mediates between the toxic gene and the host cell splicing machinery. The intron interrupts the toxic gene coding sequence and is recognized by mammalian splicing machinery but not by insect cell splicing machinery, thereby controlling the spatial and temporal expression of the toxic gene.
2Reliability
If inducible promoters are used to control toxic gene expression, then cell viability is improved, but expression control precision deteriorates
Solution Approach 1:
The patent changes the fundamental parameter of gene expression control from chemical induction (promoters) to biological recognition (splicing). By using an intron that is recognized by mammalian splicing machinery but not by insect cell machinery, the system achieves precise, automatic control of toxic gene expression based on the host cell type, eliminating the need for external induction and achieving complete suppression in non-mammalian cells.
3Productivity
If toxic genes are expressed in insect cells for vector production, then vector titer is improved, but cell death increases leading to low yields
Solution Approach 1:
The patent applies local quality by making the toxic gene sequence locally different in insect cells versus mammalian cells. The intron interrupts the coding sequence in a way that is invisible to insect cell splicing machinery (which cannot splice the intron), preserving the toxic gene's integrity in insect cells for high-yield production, while being recognized and spliced by mammalian splicing machinery in target cells, where it then exerts its toxic effect.
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 enables the production of high-titer viral vectors in insect cells while ensuring specific toxicity to mammalian cancer cells, enhancing the efficacy of suicide gene therapy for cancer treatment.
Implementation Method 1
an intron that interrupts the sequence, whereby an RNA comprising the intron can be spliced by mammalian cells but not by insect cells, to form in mammalian cells but not in insect cells, a translatable mRNA
Data Source
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AI summary
The present disclosure describes nucleic acids, and viruses comprising such nucleic acids, for growing a toxic gene in an insect cell. These nucleic acids comprise a sequence encoding a toxic polypeptide, and an intron that interrupts the sequence, whereby the intron is spliced in mammalian cells but not in insect cells. Infection of mammalian cells but not insect cells with the nucleic acids or viruses can lead to expression of toxic levels of the toxic polypeptide in mammalian cells but not in insect cells. Viruses, such as an AAV or a baculovirus comprising a nucleic acid can be grown in insect cell lines in vitro and can be administered to a subject in need of therapy, such as a subject in need of cancer therapy.