Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues.
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 alpha-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.
Gonzalez-Nicolini
Valeria
V
Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology-ETH Zurich, ETH Hoenggerberg, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
Sanchez-Bustamante
Carlota Diaz
CD
Hartenbach
Shizuka
S
Fussenegger
Martin
M
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000138:chemical synthesis*; D006801:Humans; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018085:Regulon; D025361:Streptogramins / Q000494:pharmacology; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000187:drug effects
D011994:Recombinant Proteins; D025361:Streptogramins