Vector insert-targeted integrative antisense expression system for plasmid stabilization.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• DNA vaccine and gene therapy vectors often contain toxic transgenes, leading to low yields in E. coli production hosts.
• Modifying vector sequences is undesirable for clinical products as it creates a new chemical entity.

Purpose of the Study:

• To investigate if unintended expression from spurious bacterial promoters causes insert-encoded toxicity.
• To determine if antisense RNA-mediated translation inhibition can eliminate this toxicity.
• To develop a method for improving production yields of toxic gene-encoding plasmids.

Main Methods:

• Developed the pINT PR PL vector for chromosomally integrated RNA expression.
• Expressed insert-complementary (anti-insert) RNA from a defined chromosomal site.
• Tested the system using plasmids encoding toxic retroviral gag pol and Influenza H1 hemagglutinin transgenes.

Main Results:

• Anti-insert RNA successfully eliminated leaky fluorescent protein expression.
• A fourfold increase in plasmid fermentation yield was observed for a toxic retroviral gag pol helper plasmid.
• A fourfold improvement in yield was achieved for a DNA vaccine plasmid with a toxic Influenza H1 hemagglutinin transgene.

Conclusions:

• Unintended expression of toxic proteins or peptides from alternative reading frames contributes to poor plasmid yields.
• Anti-insert chromosomal RNA expression is a versatile strategy to enhance production of plasmid vectors encoding toxic elements.
• This technology offers a general solution for improving yields without altering the therapeutic vector's sequence.