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Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression in mammalian cells and mice.

Fluri DA, Baba MD, Fussenegger M - BMC Biotechnol. (2007)

Bottom Line: Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications.To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland. david.fluri@chem.ethz.ch

ABSTRACT

Background: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features.

Results: We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.

Conclusion: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.

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AAV type 2-based regulated expression of secreted proteins. (A) Schematic representation of pDF77, harboring a SEAP expression cassette under the control of PETR, pDF143, a self-regulated expression unit expressing ET1 from PSV40 and driving SEAP from PETR and pDF109, harboring a constitutive SEAP expression unit driven by PhCMV (B) SEAP expression levels profiled 48 h after (co-)transduction of transgenic AAV particles (1000 genomic particles/cell for pDF51/77, 1000 genomic particles/cell for pDF143 and 1000 genomic particles/cell for pDF109) derived from indicated vectors and cultivated in the presence (+) or absence (-) of EM. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; ET1, erythromycin transactivator; ITR, inverted terminal repeat; pASV40, simian virus 40 polyadenylation signal; PhEF1α, human elongation factor 1α promoter; PETR, erythromycin-responsive promoter; SEAP, human placental secreted alkaline phosphatase; PSV40, simian virus 40 promoter.
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Figure 3: AAV type 2-based regulated expression of secreted proteins. (A) Schematic representation of pDF77, harboring a SEAP expression cassette under the control of PETR, pDF143, a self-regulated expression unit expressing ET1 from PSV40 and driving SEAP from PETR and pDF109, harboring a constitutive SEAP expression unit driven by PhCMV (B) SEAP expression levels profiled 48 h after (co-)transduction of transgenic AAV particles (1000 genomic particles/cell for pDF51/77, 1000 genomic particles/cell for pDF143 and 1000 genomic particles/cell for pDF109) derived from indicated vectors and cultivated in the presence (+) or absence (-) of EM. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; ET1, erythromycin transactivator; ITR, inverted terminal repeat; pASV40, simian virus 40 polyadenylation signal; PhEF1α, human elongation factor 1α promoter; PETR, erythromycin-responsive promoter; SEAP, human placental secreted alkaline phosphatase; PSV40, simian virus 40 promoter.

Mentions: Tight regulation of therapeutic transgenes remains a major challenge for current gene therapy initiatives. In order to assess whether AAV-based transduction systems enable delivery of tightly regulated expression of a human model glycoprotein, we constructed pDF77 harboring a PETR-driven SEAP (human placental secreted alkaline phosphatase) (ITR-PETR-SEAP-pASV40-ITR) (Figure 3A). Co-transduction of HT-1080 and MCF-7 with pDF77- and pDF51- (ITR-PhCMV-ET1-pASV40-ITR) derived AAV particles provided excellent regulation performance characterized by high-level SEAP production in the absence of erythromycin and repressed SEAP levels in the presence of erythromycin (Figure 3B). Furthermore, we have designed and evaluated pDF143 (ITR-PSV40-ET1-pASV40-PETR-SEAP-pASV40-ITR), a one-vector expression configuration which is isogenic to pDF141 but encoding SEAP instead of EYFP. pDF143 exhibited similar SEAP regulation profiles in HT-1080 and MCF-7 compared to co-transduction of pDF77-/pDF51-derived AAV particles suggesting that there is no interference between ET1-driving PSV40 and ET1-specific PETR (Figure 3A and 3B). Overall, maximum expression levels for the one-vector configuration were even higher than for the binary setting likely because for the binary system two different particles have to transduce a single cell which is a more rare event. Transduction of HT-1080 and MCF-7 with a constitutive control vector, pDF109 (ITR-PhCMV-intronβ-globin-SEAP-pAhgh-ITR) showed increased SEAP production compared to fully induced pDF143-derived particles, which might be associated with either higher promoter strenght or the β-globin intron.


Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression in mammalian cells and mice.

Fluri DA, Baba MD, Fussenegger M - BMC Biotechnol. (2007)

AAV type 2-based regulated expression of secreted proteins. (A) Schematic representation of pDF77, harboring a SEAP expression cassette under the control of PETR, pDF143, a self-regulated expression unit expressing ET1 from PSV40 and driving SEAP from PETR and pDF109, harboring a constitutive SEAP expression unit driven by PhCMV (B) SEAP expression levels profiled 48 h after (co-)transduction of transgenic AAV particles (1000 genomic particles/cell for pDF51/77, 1000 genomic particles/cell for pDF143 and 1000 genomic particles/cell for pDF109) derived from indicated vectors and cultivated in the presence (+) or absence (-) of EM. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; ET1, erythromycin transactivator; ITR, inverted terminal repeat; pASV40, simian virus 40 polyadenylation signal; PhEF1α, human elongation factor 1α promoter; PETR, erythromycin-responsive promoter; SEAP, human placental secreted alkaline phosphatase; PSV40, simian virus 40 promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211474&req=5

Figure 3: AAV type 2-based regulated expression of secreted proteins. (A) Schematic representation of pDF77, harboring a SEAP expression cassette under the control of PETR, pDF143, a self-regulated expression unit expressing ET1 from PSV40 and driving SEAP from PETR and pDF109, harboring a constitutive SEAP expression unit driven by PhCMV (B) SEAP expression levels profiled 48 h after (co-)transduction of transgenic AAV particles (1000 genomic particles/cell for pDF51/77, 1000 genomic particles/cell for pDF143 and 1000 genomic particles/cell for pDF109) derived from indicated vectors and cultivated in the presence (+) or absence (-) of EM. SEAP expression is shown in units/liter (U/l) as defined by Schlatter et al. [52]. Abbreviations: EM, erythromycin; ET1, erythromycin transactivator; ITR, inverted terminal repeat; pASV40, simian virus 40 polyadenylation signal; PhEF1α, human elongation factor 1α promoter; PETR, erythromycin-responsive promoter; SEAP, human placental secreted alkaline phosphatase; PSV40, simian virus 40 promoter.
Mentions: Tight regulation of therapeutic transgenes remains a major challenge for current gene therapy initiatives. In order to assess whether AAV-based transduction systems enable delivery of tightly regulated expression of a human model glycoprotein, we constructed pDF77 harboring a PETR-driven SEAP (human placental secreted alkaline phosphatase) (ITR-PETR-SEAP-pASV40-ITR) (Figure 3A). Co-transduction of HT-1080 and MCF-7 with pDF77- and pDF51- (ITR-PhCMV-ET1-pASV40-ITR) derived AAV particles provided excellent regulation performance characterized by high-level SEAP production in the absence of erythromycin and repressed SEAP levels in the presence of erythromycin (Figure 3B). Furthermore, we have designed and evaluated pDF143 (ITR-PSV40-ET1-pASV40-PETR-SEAP-pASV40-ITR), a one-vector expression configuration which is isogenic to pDF141 but encoding SEAP instead of EYFP. pDF143 exhibited similar SEAP regulation profiles in HT-1080 and MCF-7 compared to co-transduction of pDF77-/pDF51-derived AAV particles suggesting that there is no interference between ET1-driving PSV40 and ET1-specific PETR (Figure 3A and 3B). Overall, maximum expression levels for the one-vector configuration were even higher than for the binary setting likely because for the binary system two different particles have to transduce a single cell which is a more rare event. Transduction of HT-1080 and MCF-7 with a constitutive control vector, pDF109 (ITR-PhCMV-intronβ-globin-SEAP-pAhgh-ITR) showed increased SEAP production compared to fully induced pDF143-derived particles, which might be associated with either higher promoter strenght or the β-globin intron.

Bottom Line: Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications.To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland. david.fluri@chem.ethz.ch

ABSTRACT

Background: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features.

Results: We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression.

Conclusion: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.

Show MeSH
Related in: MedlinePlus