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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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Self- and auto-regulated AAV type 2-based expression of fluorescent proteins. (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pAI, synthetic polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PHSP70min, minimal version of the Drosophila melanogaster heat-shock protein 70 promoter; PSV40, simian virus 40 promoter.
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Figure 2: Self- and auto-regulated AAV type 2-based expression of fluorescent proteins. (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pAI, synthetic polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PHSP70min, minimal version of the Drosophila melanogaster heat-shock protein 70 promoter; PSV40, simian virus 40 promoter.

Mentions: In order to enable delivery of macrolide-responsive transgene expression in a most compact format and a single transduction event we engineered expression of the macrolide-dependent transactivator ET1 and the desired target gene into a single AAV vector configuration. The pioneering one-vector design concept consisted of tandem constitutive ET1 (PSV40-ET1-pASV40) and macrolide-responsive EYFP expression units (PETR-EYFP-pASV40) placed between two ITRs (Figure 2A). pDF141- (ITR-PSV40-ET1-pASV40-PETR-EYFP-pASV40-ITR) derived AAV particles efficiently transduced HT-1080 and MCF-7 and mediated high-level EYFP expression when transgenic populations were grown for 48 h in the absence of erythromycin (Figure 2B). To prevent any deregulation of PETR by promoters or enhancers encoded in cis and to minimize the overall size of the transgene unit, we assembled (i) pDF124 (ITR-PETR-EYFP-IRESEMCV-ET1-pASV40-ITR) a dicistronic autoregulated PETR-driven AAV expression configuration harboring an ITR-flanked PETR-driven dicistronic expression cassette with EYFP in the first and ET1 in the second cistron separated by a internal ribosome entry site of encephalomyocarditisviral origin (IRESEMCV) and (ii) pDF89 (ITR-pAI-EYFP←PhCMVmin-ETR-PHSP70min→ET1-pASV40-ITR) containing a bidirectional expression unit consisting of a central ET1-specific operator ETR flanked by PETR driving EYFP expression in one direction and a PHSP70min (minimal version of the Drosophila melanogaster heat-shock protein 70 promoter) driving ET1 in the opposite direction.


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

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

Self- and auto-regulated AAV type 2-based expression of fluorescent proteins. (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pAI, synthetic polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PHSP70min, minimal version of the Drosophila melanogaster heat-shock protein 70 promoter; PSV40, simian virus 40 promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Self- and auto-regulated AAV type 2-based expression of fluorescent proteins. (A) Schematic representation of an auto-regulated (pDF124), a bidirectional (pDF89) and a self-regulated (pDF141) AAV type 2-based expression unit. (B) Fluorescence micrographs of human fibrosarcoma cells (HT-1080) and a human breast cancer cell line (MCF-7) transduced with pDF124-, pDF89- and pDF141-derived AAV particles (2000 genomic particles/cell) cultivated in the presence (+) and absence (-) of EM. (C) FACS-mediated quantification of EYFP in HT-1080 transduced with equal amounts of viral particles (2000 genomic particles/cell) and cultivated in the presence (+EM) and absence (-EM) of erythromycin. Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; ETR, ET1-specific operator; EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; ITR, inverted terminal repeat; pAI, synthetic polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PHSP70min, minimal version of the Drosophila melanogaster heat-shock protein 70 promoter; PSV40, simian virus 40 promoter.
Mentions: In order to enable delivery of macrolide-responsive transgene expression in a most compact format and a single transduction event we engineered expression of the macrolide-dependent transactivator ET1 and the desired target gene into a single AAV vector configuration. The pioneering one-vector design concept consisted of tandem constitutive ET1 (PSV40-ET1-pASV40) and macrolide-responsive EYFP expression units (PETR-EYFP-pASV40) placed between two ITRs (Figure 2A). pDF141- (ITR-PSV40-ET1-pASV40-PETR-EYFP-pASV40-ITR) derived AAV particles efficiently transduced HT-1080 and MCF-7 and mediated high-level EYFP expression when transgenic populations were grown for 48 h in the absence of erythromycin (Figure 2B). To prevent any deregulation of PETR by promoters or enhancers encoded in cis and to minimize the overall size of the transgene unit, we assembled (i) pDF124 (ITR-PETR-EYFP-IRESEMCV-ET1-pASV40-ITR) a dicistronic autoregulated PETR-driven AAV expression configuration harboring an ITR-flanked PETR-driven dicistronic expression cassette with EYFP in the first and ET1 in the second cistron separated by a internal ribosome entry site of encephalomyocarditisviral origin (IRESEMCV) and (ii) pDF89 (ITR-pAI-EYFP←PhCMVmin-ETR-PHSP70min→ET1-pASV40-ITR) containing a bidirectional expression unit consisting of a central ET1-specific operator ETR flanked by PETR driving EYFP expression in one direction and a PHSP70min (minimal version of the Drosophila melanogaster heat-shock protein 70 promoter) driving ET1 in the opposite direction.

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