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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 erythromycin-inducible EYFP expression. (A) Schematic representation of pDF126 harboring a PhCMV-driven E-KRAB expression unit and pDF206/207/208 encoding EYFP transcribed by PETRON promoter derivatives containing 8 (pDF207), 4 (pDF208) and 2 (pDF209) E-KRAB-specific ETR operator modules. (B) Fluorescence micrographs of HT-1080 and MCF-7 co-transduced with pDF209/pDF126-derived AAV particles (1000 genomic particles/cell for pDF207/208/209 and 4000 genomic particles/cell for pDF126) and cultivated in the presence (+EM) and absence (-EM) of erythromycin (C) FACS-mediated quantification of EYFP expression in HT-1080. EYFP-specific FACS diagrams of HT-1080 co-transduced (4000 and 1000 genomic particles/cell) by pDF126/pDF207, pDF126/pDF208 and pDF126/pDF209 were cultivated for 48 h in the presence (+) and absence (-) of EM. Abbreviations: EM, erythromycin; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; KRAB, kruppel associated box; PETRON, erythromycin-inducible promoter.
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Figure 4: AAV type 2-based erythromycin-inducible EYFP expression. (A) Schematic representation of pDF126 harboring a PhCMV-driven E-KRAB expression unit and pDF206/207/208 encoding EYFP transcribed by PETRON promoter derivatives containing 8 (pDF207), 4 (pDF208) and 2 (pDF209) E-KRAB-specific ETR operator modules. (B) Fluorescence micrographs of HT-1080 and MCF-7 co-transduced with pDF209/pDF126-derived AAV particles (1000 genomic particles/cell for pDF207/208/209 and 4000 genomic particles/cell for pDF126) and cultivated in the presence (+EM) and absence (-EM) of erythromycin (C) FACS-mediated quantification of EYFP expression in HT-1080. EYFP-specific FACS diagrams of HT-1080 co-transduced (4000 and 1000 genomic particles/cell) by pDF126/pDF207, pDF126/pDF208 and pDF126/pDF209 were cultivated for 48 h in the presence (+) and absence (-) of EM. Abbreviations: EM, erythromycin; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; KRAB, kruppel associated box; PETRON, erythromycin-inducible promoter.

Mentions: Various therapeutic initiatives require transgene control systems, which trigger heterologous target genes after administration of the regulating agent and remain repressed in the absence of the inducer [39]. We have constructed binary EON-controlled AAV-based expression systems consisting of pDF126 (ITR-PhCMV-Intron-β-globin-E-KRAB-pAhgh-ITR), and pDF207 (ITR-PETRON8-EYFP-pASV40-ITR), pDF208 (ITR-PETRON4-EYFP-pASV40-ITR) or pDF209 (ITR-PETRON2-EYFP-pASV40-ITR), which harbor EYFP under control of different E-KRAB-specific macrolide-inducible promoters characterized by different ETR tandem repeats placed 3' of the constitutive PSV40 promoter [14]. Functionality of the ON-type system was validated by co-transduction of pDF126/pDF209-derived into HT-1080 and MCF-7 and profiling of EYFP expression after 48 h cultivation in the presence or absence of erythromycin (Figure 4B). FACS-based comparison of HT-1080 co-transduced with pDF126-/pDF207-, pDF126-/pDF208- and pDF126-/pDF209-derived AAV particles and cultivated for 48 h in the presence and absence of erythromycin revealed that although overall induction factors were almost identical among different particle combinations, basal and maximum expression levels were a function of the number of ETR repeats associated with PSV40 (Figures 4A and 4C).


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 erythromycin-inducible EYFP expression. (A) Schematic representation of pDF126 harboring a PhCMV-driven E-KRAB expression unit and pDF206/207/208 encoding EYFP transcribed by PETRON promoter derivatives containing 8 (pDF207), 4 (pDF208) and 2 (pDF209) E-KRAB-specific ETR operator modules. (B) Fluorescence micrographs of HT-1080 and MCF-7 co-transduced with pDF209/pDF126-derived AAV particles (1000 genomic particles/cell for pDF207/208/209 and 4000 genomic particles/cell for pDF126) and cultivated in the presence (+EM) and absence (-EM) of erythromycin (C) FACS-mediated quantification of EYFP expression in HT-1080. EYFP-specific FACS diagrams of HT-1080 co-transduced (4000 and 1000 genomic particles/cell) by pDF126/pDF207, pDF126/pDF208 and pDF126/pDF209 were cultivated for 48 h in the presence (+) and absence (-) of EM. Abbreviations: EM, erythromycin; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; KRAB, kruppel associated box; PETRON, erythromycin-inducible promoter.
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Figure 4: AAV type 2-based erythromycin-inducible EYFP expression. (A) Schematic representation of pDF126 harboring a PhCMV-driven E-KRAB expression unit and pDF206/207/208 encoding EYFP transcribed by PETRON promoter derivatives containing 8 (pDF207), 4 (pDF208) and 2 (pDF209) E-KRAB-specific ETR operator modules. (B) Fluorescence micrographs of HT-1080 and MCF-7 co-transduced with pDF209/pDF126-derived AAV particles (1000 genomic particles/cell for pDF207/208/209 and 4000 genomic particles/cell for pDF126) and cultivated in the presence (+EM) and absence (-EM) of erythromycin (C) FACS-mediated quantification of EYFP expression in HT-1080. EYFP-specific FACS diagrams of HT-1080 co-transduced (4000 and 1000 genomic particles/cell) by pDF126/pDF207, pDF126/pDF208 and pDF126/pDF209 were cultivated for 48 h in the presence (+) and absence (-) of EM. Abbreviations: EM, erythromycin; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; KRAB, kruppel associated box; PETRON, erythromycin-inducible promoter.
Mentions: Various therapeutic initiatives require transgene control systems, which trigger heterologous target genes after administration of the regulating agent and remain repressed in the absence of the inducer [39]. We have constructed binary EON-controlled AAV-based expression systems consisting of pDF126 (ITR-PhCMV-Intron-β-globin-E-KRAB-pAhgh-ITR), and pDF207 (ITR-PETRON8-EYFP-pASV40-ITR), pDF208 (ITR-PETRON4-EYFP-pASV40-ITR) or pDF209 (ITR-PETRON2-EYFP-pASV40-ITR), which harbor EYFP under control of different E-KRAB-specific macrolide-inducible promoters characterized by different ETR tandem repeats placed 3' of the constitutive PSV40 promoter [14]. Functionality of the ON-type system was validated by co-transduction of pDF126/pDF209-derived into HT-1080 and MCF-7 and profiling of EYFP expression after 48 h cultivation in the presence or absence of erythromycin (Figure 4B). FACS-based comparison of HT-1080 co-transduced with pDF126-/pDF207-, pDF126-/pDF208- and pDF126-/pDF209-derived AAV particles and cultivated for 48 h in the presence and absence of erythromycin revealed that although overall induction factors were almost identical among different particle combinations, basal and maximum expression levels were a function of the number of ETR repeats associated with PSV40 (Figures 4A and 4C).

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