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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 macrolide-responsive EYFP expression. (A) Schematic representation of AAV type 2-based vectors encoding ET1 under control of the constitutive PhCMV promoter (pDF51) and EYFP driven by PETR (pDF54) or a constitutive hCMV promoter (pDF60). (B) Fluorescence micrographs of CHO-K1, HT-1080, NHDF and MCF-7 either co-transduced with pDF51/pDF54 and cultivated in the presence (+) and absence (-) of erythromycin (EM) or transduced with pDF60 (1000 genomic particles/cell for each vector). Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; pAhgh, human growth hormone polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PhCMV, human immediate early cytomegalovirus promoter; Selected restriction sites: A, AccI; As, AscI; Bg, BglII; Bs, BstBI; Bsa, BsaBI; C, ClaI; E, EcoRI; H, HindIII; Hi, HincII; M, MluI; N, NdeI; Nh, NheI; Nr, NruI; P, PacI; Pm, PmeI; Pml, PmlI; S, SalI; Sa, SacII Sp, SpeI; Sph, SphI; Sw, SwaI; X, XbaI.
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Figure 1: AAV type 2-based macrolide-responsive EYFP expression. (A) Schematic representation of AAV type 2-based vectors encoding ET1 under control of the constitutive PhCMV promoter (pDF51) and EYFP driven by PETR (pDF54) or a constitutive hCMV promoter (pDF60). (B) Fluorescence micrographs of CHO-K1, HT-1080, NHDF and MCF-7 either co-transduced with pDF51/pDF54 and cultivated in the presence (+) and absence (-) of erythromycin (EM) or transduced with pDF60 (1000 genomic particles/cell for each vector). Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; pAhgh, human growth hormone polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PhCMV, human immediate early cytomegalovirus promoter; Selected restriction sites: A, AccI; As, AscI; Bg, BglII; Bs, BstBI; Bsa, BsaBI; C, ClaI; E, EcoRI; H, HindIII; Hi, HincII; M, MluI; N, NdeI; Nh, NheI; Nr, NruI; P, PacI; Pm, PmeI; Pml, PmlI; S, SalI; Sa, SacII Sp, SpeI; Sph, SphI; Sw, SwaI; X, XbaI.

Mentions: We have engineered serotype 2-based adeno-associated viral particles for transduction of macrolide-responsive expression of the enhanced yellow fluorescent protein (EYFP). The generic design consisted of a set of two vectors: pDF51 (ITR-PhCMV-intronβ-globin-ET1-pAHGH-ITR) harboring an ITR (inverted terminal repeats)-flanked PhCMV- (human cytomegalovirus immediate early promoter) driven and pAhgh- (human growth hormone-derived polyadenylation site) terminated ET1 (macrolide-dependent transactivator) expression unit containing a β-globin intron (intronβ-globin) reported to increase transcript processing and overall ET1 production levels and pDF54 (ITR-PETR-EYFP-pASV40-ITR) containing an ITR- flanked expression unit encoding PETR- (macrolide-responsive promoter) driven and pASV40- (simian virus 40-derived polyadenylation site) terminated EYFP (enhanced yellow fluorescent protein-encoding gene) expression cassette (Figure 1A). Transgenic AAV-derived particles produced by transient co-transfection of either pDF60, pDF51 or pDF54 with the helper construct pDG providing constitutive processing and assembly functions (adenovirus E2A, E4 and VA as well as AAV rep and cap genes; [38]) in trans into HEK293-T were validated for regulated EYFP expression by co-transduction of pDF51- and pDF54-derived AAV particles into representative cell types such as Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary normal human dermal fibroblasts (NHDF) and human breast cancer cells (MCF-7) cultivated in the presence (+EM) or absence (-EM) of the macrolide antibiotic erythromycin (EM). Fluorescence micrographs of transduced populations maintained for 48 h in erythromycin-free medium revealed bright green fluorescence in all cell types. Transduced populations cultivated for two days in the presence of 1 μg/ml erythromycin showed no important EYFP expression. As a positive control all cell types were transduced with AAV particles harboring EYFP driven by a constitutive human cytomegolovirus immediate early promoter (PhCMV) (Figure 1B).


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 macrolide-responsive EYFP expression. (A) Schematic representation of AAV type 2-based vectors encoding ET1 under control of the constitutive PhCMV promoter (pDF51) and EYFP driven by PETR (pDF54) or a constitutive hCMV promoter (pDF60). (B) Fluorescence micrographs of CHO-K1, HT-1080, NHDF and MCF-7 either co-transduced with pDF51/pDF54 and cultivated in the presence (+) and absence (-) of erythromycin (EM) or transduced with pDF60 (1000 genomic particles/cell for each vector). Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; pAhgh, human growth hormone polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PhCMV, human immediate early cytomegalovirus promoter; Selected restriction sites: A, AccI; As, AscI; Bg, BglII; Bs, BstBI; Bsa, BsaBI; C, ClaI; E, EcoRI; H, HindIII; Hi, HincII; M, MluI; N, NdeI; Nh, NheI; Nr, NruI; P, PacI; Pm, PmeI; Pml, PmlI; S, SalI; Sa, SacII Sp, SpeI; Sph, SphI; Sw, SwaI; X, XbaI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: AAV type 2-based macrolide-responsive EYFP expression. (A) Schematic representation of AAV type 2-based vectors encoding ET1 under control of the constitutive PhCMV promoter (pDF51) and EYFP driven by PETR (pDF54) or a constitutive hCMV promoter (pDF60). (B) Fluorescence micrographs of CHO-K1, HT-1080, NHDF and MCF-7 either co-transduced with pDF51/pDF54 and cultivated in the presence (+) and absence (-) of erythromycin (EM) or transduced with pDF60 (1000 genomic particles/cell for each vector). Abbreviations: EM, erythromycin; ET1, erythromycin-dependent transactivator; EYFP, enhanced yellow fluorescent protein; ITR, inverted terminal repeat; pAhgh, human growth hormone polyadenylation signal; pASV40, simian virus 40 polyadenylation signal; PETR, erythromycin-responsive promoter; PhCMV, human immediate early cytomegalovirus promoter; Selected restriction sites: A, AccI; As, AscI; Bg, BglII; Bs, BstBI; Bsa, BsaBI; C, ClaI; E, EcoRI; H, HindIII; Hi, HincII; M, MluI; N, NdeI; Nh, NheI; Nr, NruI; P, PacI; Pm, PmeI; Pml, PmlI; S, SalI; Sa, SacII Sp, SpeI; Sph, SphI; Sw, SwaI; X, XbaI.
Mentions: We have engineered serotype 2-based adeno-associated viral particles for transduction of macrolide-responsive expression of the enhanced yellow fluorescent protein (EYFP). The generic design consisted of a set of two vectors: pDF51 (ITR-PhCMV-intronβ-globin-ET1-pAHGH-ITR) harboring an ITR (inverted terminal repeats)-flanked PhCMV- (human cytomegalovirus immediate early promoter) driven and pAhgh- (human growth hormone-derived polyadenylation site) terminated ET1 (macrolide-dependent transactivator) expression unit containing a β-globin intron (intronβ-globin) reported to increase transcript processing and overall ET1 production levels and pDF54 (ITR-PETR-EYFP-pASV40-ITR) containing an ITR- flanked expression unit encoding PETR- (macrolide-responsive promoter) driven and pASV40- (simian virus 40-derived polyadenylation site) terminated EYFP (enhanced yellow fluorescent protein-encoding gene) expression cassette (Figure 1A). Transgenic AAV-derived particles produced by transient co-transfection of either pDF60, pDF51 or pDF54 with the helper construct pDG providing constitutive processing and assembly functions (adenovirus E2A, E4 and VA as well as AAV rep and cap genes; [38]) in trans into HEK293-T were validated for regulated EYFP expression by co-transduction of pDF51- and pDF54-derived AAV particles into representative cell types such as Chinese hamster ovary cells (CHO-K1), human fibrosarcoma cells (HT-1080), primary normal human dermal fibroblasts (NHDF) and human breast cancer cells (MCF-7) cultivated in the presence (+EM) or absence (-EM) of the macrolide antibiotic erythromycin (EM). Fluorescence micrographs of transduced populations maintained for 48 h in erythromycin-free medium revealed bright green fluorescence in all cell types. Transduced populations cultivated for two days in the presence of 1 μg/ml erythromycin showed no important EYFP expression. As a positive control all cell types were transduced with AAV particles harboring EYFP driven by a constitutive human cytomegolovirus immediate early promoter (PhCMV) (Figure 1B).

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