A single adenovirus vector mediates doxycycline-controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors

Abstract

Ex vivo gene transfer is emerging as a promising therapeutic approach to human neurodegenerative diseases. By combining efficient methodologies for cell amplification and gene delivery, large numbers of cells can be generated with the capacity to synthesize therapeutic molecules. These cells can then be transplanted into the degenerating central nervous system (CNS). Applying this approach to human diseases will require the development of suitable cellular vehicles, as well as safe gene delivery systems capable of tightly controlled transgene expression. For such brain repair technologies, human neural progenitors may be extremely valuable, because of their human CNS origin and developmental potential. We have used these cells to develop a system for the regulated expression of a gene of therapeutic potential. We report the construction of a single adenovirus encoding human tyrosine hydroxylase 1 (hTH-1) under the negative control of the tetracycline-based gene regulatory system. Human neural progenitors infected with this vector produced large amounts of hTH-1. Most importantly, doxycycline allowed a reversible switch of transgene transcription both in vitro and in vivo. This system may be applied to the development of therapies for human neurodegenerative diseases.

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Figure 1: AdPGK♦tet♦hTH-1.
Figure 2: (A) Infection of human neural progenitors with AdPGK♦tet♦hTH-1 at various MOIs.
Figure 3: Kinetics of clearance (A) and resumption (B) of hTH activity from cultures infected with AdPGK♦tet♦hTH-1 (MOI = 40) after addition (A) or withdrawal (B) of doxycycline (5 ng/ml) on day 0 (5.5 days after infection).
Figure 4: TH immunoreactivity (brown immunoperoxidase staining) in intrastriatal grafts of human neural progenitors infected with AdPGK♦tet♦hTH-1 (MOI = 40).
Figure 5: TH expression in grafts of human neural progenitors infected with AdPGK♦tet♦hTH-1 (MOI = 40), as determined by anti-TH immunohistochemistry (brown immunoperoxidase staining) and in situ hybridization using a [35S]-labeled hTH-1 specific antisense oligonucleotide (black silver grains) on 15 μm cryostat brain sections from (A) an untreated animal and (B) an animal treated daily with 1 mg/ml doxycycline.
Figure 6: TH expression in grafts of human neural progenitors infected with AdPGK♦tet♦hTH-1 as determined by immunohistochemistry (A–D) or in situ hybridization using an oligonucleotide probe labeled with digoxygenin-tagged rUTP (E–G).

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Acknowledgements

We thank Gilles Pitiot for helpful discussions and critical review of the manuscript. We also thank Amelia Sanchez-Capelo for her unremitting contribution to the construction of AdPGK♦tet♦hTH-1. We thank Novartis for generously providing cyclosporine A. This work was supported by grants from the Association Francaise contre les Myopathies, the Centre National de la Recherche Scientifique, the Conséil Régional d'Ile de France, EC contract No. 951012, the European Science Foundation, the Institut pour la Recherche sur la Moelle Epiniere and Retina France.

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Correspondence to Jacques Mallet.

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Corti, O., Sabaté, O., Horellou, P. et al. A single adenovirus vector mediates doxycycline-controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors. Nat Biotechnol 17, 349–354 (1999). https://doi.org/10.1038/7901

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