Short regulatory DNA sequences to target brain endothelial cells for gene therapy

Hanna Graßhoff; Helge Müller-Fielitz; Godwin K. Dogbevia; Jakob Körbelin; Jacqueline Bannach; Carl M.G. Vahldieck; Kristina Kusche-Vihrog; Olaf Jöhren; Oliver J. Müller; Ruben Nogueiras; Vincent Prevot; Markus Schwaninger

doi:10.1177/0271678X211039617

Short regulatory DNA sequences to target brain endothelial cells for gene therapy

Hanna Graßhoff, Helge Müller-Fielitz, Godwin K. Dogbevia, Jakob Körbelin, Jacqueline Bannach, Carl M.G. Vahldieck, Kristina Kusche-Vihrog, Olaf Jöhren, Oliver J. Müller, Ruben Nogueiras, Vincent Prevot, Markus Schwaninger^*

^*Corresponding author for this work

14 Citations (Scopus)

Abstract

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

Original language	English
Journal	Journal of Cerebral Blood Flow and Metabolism
Volume	42
Issue number	1
Pages (from-to)	104-120
Number of pages	17
ISSN	0271-678X
DOIs	https://doi.org/10.1177/0271678X211039617
Publication status	Published - 01.2022

Funding

We are grateful to Dr. Rolf Sprengel, University of Heidelberg, Germany, for providing the plasmid p179, to Dr. Mazahir Hasan, Achucarro Basque Center for Neuroscience, Spain, for providing the plasmid pAAV-CAG-iCre-2A-eGFP, to Dr. Sebastian K?gler, University Medical Center G?ttingen, Germany, for providing the plasmid p-6p1 (hSYN -eGFP-W), and to Dr. Urban Deutsch, University of Bern, Switzerland, for advice on the short Tie2 intron. We also thank Frauke Spiecker, Wiebke Brandt, Ines St?lting, and Christine Eichholz, University of L?beck, for expert technical support. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the European Research Council (ERC) Synergy Grant-2019-WATCH-810331 to VP, RN, and MS and by grants of the Deutsche Forschungsgemeinschaft (DFG, SCHW 416/5-3, 9-1). The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the European Research Council (ERC) Synergy Grant-2019-WATCH-810331 to VP, RN, and MS and by grants of the Deutsche Forschungsgemeinschaft (DFG, SCHW 416/5-3, 9-1). Acknowledgements

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Research Areas and Centers

Academic Focus: Center for Brain, Behavior and Metabolism (CBBM)

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Cite this

Graßhoff, H., Müller-Fielitz, H., Dogbevia, G. K., Körbelin, J., Bannach, J., Vahldieck, C. M. G., Kusche-Vihrog, K., Jöhren, O., Müller, O. J., Nogueiras, R., Prevot, V., & Schwaninger, M. (2022). Short regulatory DNA sequences to target brain endothelial cells for gene therapy. Journal of Cerebral Blood Flow and Metabolism, 42(1), 104-120. https://doi.org/10.1177/0271678X211039617

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title = "Short regulatory DNA sequences to target brain endothelial cells for gene therapy",

abstract = "Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.",

author = "Hanna Gra{\ss}hoff and Helge M{\"u}ller-Fielitz and Dogbevia, \{Godwin K.\} and Jakob K{\"o}rbelin and Jacqueline Bannach and Vahldieck, \{Carl M.G.\} and Kristina Kusche-Vihrog and Olaf J{\"o}hren and M{\"u}ller, \{Oliver J.\} and Ruben Nogueiras and Vincent Prevot and Markus Schwaninger",

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Graßhoff, H, Müller-Fielitz, H, Dogbevia, GK, Körbelin, J, Bannach, J, Vahldieck, CMG, Kusche-Vihrog, K , Jöhren, O, Müller, OJ, Nogueiras, R, Prevot, V & Schwaninger, M 2022, 'Short regulatory DNA sequences to target brain endothelial cells for gene therapy', Journal of Cerebral Blood Flow and Metabolism, vol. 42, no. 1, pp. 104-120. https://doi.org/10.1177/0271678X211039617

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T1 - Short regulatory DNA sequences to target brain endothelial cells for gene therapy

AU - Graßhoff, Hanna

AU - Müller-Fielitz, Helge

AU - Dogbevia, Godwin K.

AU - Körbelin, Jakob

AU - Bannach, Jacqueline

AU - Vahldieck, Carl M.G.

AU - Kusche-Vihrog, Kristina

AU - Jöhren, Olaf

AU - Müller, Oliver J.

AU - Nogueiras, Ruben

AU - Prevot, Vincent

AU - Schwaninger, Markus

PY - 2022/1

Y1 - 2022/1

N2 - Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

AB - Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

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Short regulatory DNA sequences to target brain endothelial cells for gene therapy

Abstract

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Research Areas and Centers

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