Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues

✍ Scribed by Valeria Gonzalez-Nicolini; Carlota Diaz Sanchez-Bustamante; Shizuka Hartenbach; Martin Fussenegger

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 224 KB
• Volume: 8
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.960

✦ Synopsis

Abstract

Background

Adenoviral particles can efficiently transduce a broad spectrum of cell types, so they are widely used in basic research and clinical trials.

Methods

We have developed a novel adenoviral vector platform for delivery of constitutive or streptogramin‐inducible expression of up to three therapeutic transgenes into a variety of murine and human cell lines, primary cells and microtissues.

Results

Coordinated expression of three independent transgenes in a compact genetic format was achieved by two different expression configurations: (i) The multicistronic expression format consisting of a single constitutive (simian virus 40 promoter, P~SV40~; murine or human cytomegalovirus immediate‐early promoter, P~mCMV~, P~hCMV~) or regulated (streptogramin‐inducible) promoters (P~PIR~ON2) driving the expression of a single multicistronic transcript of which the first cistron is translated in a cap‐dependent manner and the two subsequent ones by internal ribosome entry site (IRES)‐mediated translation initiation. (ii) The triple‐transcript expression configuration, in which a combination of well‐established (P~SV40~, P~hCMV~, P~mCMV~) and novel synthetic constitutive promoters (P~GTX~) control transcription of three expression units. The constitutive multigene expression design enabled coordinated high‐level expression of the Bacillus stearothermophilus‐derived secreted α‐amylase (SAMY), the human vascular endothelial growth factor 121 (VEGF~121~) and the human placental secreted alkaline phosphatase (SEAP) in monolayer populations and microtissues of Chinese hamster ovary cells (CHO‐K1), human fibrosarcoma cells (HT‐1080), primary neonatal rat cardiomyocytes (NRCs) and primary human aortic fibroblasts (HAFs). Streptogramin‐inducible tricistronic SAMY‐VEGF~121~‐SEAP expression provided excellent regulation performance—high‐level induction in the presence of the streptogramin antibiotic pristinamycin I (PI), near‐undetectable basal expression in the absence of PI, optimal adjustability and perfect reversibility—in all cell types, in particular in NRCs and NRC‐derived myocardial microtissues.

Conclusions

Triple‐transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size‐constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios. Copyright © 2006 John Wiley & Sons, Ltd.