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Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures

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Abstract

Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIRES-hrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 μg of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood–Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis.

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Acknowledgments

The authors thank Minerva Crespo-Ramírez from Division of Neuroscience, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, and Isaías Angulo Viurcos from Unidad de Investigación Biomédica en Infectología e Inmunología, Hospital de Infectología, Centro Médico Nacional “La Raza”, IMSS for their technical support and MDV Enrique García-Formenti and MDV Jaime Herrera Pérez from the animal facility at Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social for their excellent medical and technical assistance.

Funding

This work was funded by grants from the Fondo de InvestigaciónenSalud (IMSS, Number: FIS/IMSS/PROT/G14/1320) and (FIS/IMSS/PROT/PRIO/18/071).

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Correspondence to Emiliano Tesoro-Cruz or Norma Oviedo.

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All authors declare that there is no actual or potential conflict of interest.

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Tesoro-Cruz, E., Oviedo, N., Manuel-Apolinar, L. et al. Ophthalmic Administration of a DNA Plasmid Harboring the Murine Tph2 Gene: Evidence of Recombinant Tph2-FLAG in Brain Structures. Mol Biotechnol 62, 200–209 (2020). https://doi.org/10.1007/s12033-020-00239-x

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Keywords

  • Ophthalmic administration
  • Tph2 gene
  • Serotonin
  • Brainstem
  • Depression