New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable...

Area of Science:

• Molecular Biology
• Gene Therapy Vector Design

Background:

• Multicistronic expression systems are crucial for efficient gene delivery.
• Existing vectors often face limitations in size, flexibility, and integration efficiency.

Purpose of the Study:

• To design and characterize novel pTRIDENT vectors for coordinated multicistronic expression.
• To enable flexible transgene cassette swapping and integration into lentiviral vectors.

Main Methods:

• Engineered pTRIDENT vectors utilizing small, efficient internal ribosome entry sites (GTX or Rbm3 type).
• Incorporated homing endonuclease sites for promoter/expression unit/polyadenylation site swapping.
• Integrated pTRIDENT cassettes into HIV-1-based lentiviral vectors.
• Quantified multicistronic expression profiles in CHO-K1, NIH/3T3, and HT-1080 cell lines.

Main Results:

• Demonstrated coordinated expression of transgenes including urokinase-type plasminogen activator (u-PA(LMW)), alpha-amylase (SAMY), vascular endothelial growth factor (hVEGF), and placental alkaline phosphatase (SEAP).
• Achieved size-optimized vectors with high transgene expression efficiency.
• Successfully transduced primary human chondrocytes with lentiviral particles containing pTRIDENT-derived expression cassettes.

Conclusions:

• pTRIDENT vectors offer a versatile and efficient platform for multicistronic gene expression.
• The system facilitates seamless integration into lentiviral vectors for advanced gene therapy applications.
• Validated the efficacy of pTRIDENT vectors in various cell types, including primary human cells.