D-TAT transporter as an ocular peptide delivery system.
BACKGROUND
Future treatment for genetic diseases may involve the replacement of malfunctioning genes through virus-mediated gene therapy. However, this approach is plagued with many problems, both ethical and scientific. Therefore, alternative treatments based on new molecules may represent a safer option. Molecular treatment of many eye diseases will need to bring active molecules into the photoreceptors. Recently, the trans-activator protein (TAT) human immunodeficiency virus type 1 (HIV-1) transcriptional factor has proven to be effective in transporting molecules across cellular membranes. The half-life of these molecules does not exceed 48 hours. The potential use of the retro-inverso form of the TAT (D-TAT) peptide, the protein transducing domain of the HIV-1 transcriptional factor, as a molecular transporter was investigated.


METHODS
FITC-labelled D-TAT (D-TAT FITC) was applied to the 661W murine photoreceptor cell line in culture. The labelled peptide was also injected into the vitreous body or the subretinal space of adult mice. Cells and cryosections of eyes were analysed under fluorescence microscopy at various time points after peptide treatment. Coimmunostaining with various antibodies was performed in order to characterize the transduces cells.


RESULTS
D-TAT was effective in transducing photoreceptor cells in culture. Transduction of D-TAT FITC was also effective when injected into the vitreous or subretinal space and was observed for a longer period of time than L-TAT FITC.


CONCLUSIONS
The retro-inverso form of the TAT sequence is effective in transducing cells from various compartments of the eye. After 14 days, the D-TAT FITC was clearly visible in the retina whereas L-TAT FITC had almost disappeared. The D-TAT peptide represents an interesting molecular transporter that, when coupled to a specific effector, may have potential therapeutic future, especially when a long-lasting action is needed.
Schorderet
Daniel F
DF
IRO-Institut de Recherche en Ophtalmologie, Sion, Switzerland. daniel.schorderet@iro.vsnet.ch
Manzi
Violaine d'Allèves
Vd
Canola
Kriss
K
Bonny
Christophe
C
Arsenijevic
Yvan
Y
Munier
Francis L
FL
Maurer
Fabienne
F
D000818:Animals; D002478:Cells, Cultured; D016503:Drug Delivery Systems; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D007267:Injections; D051379:Mice; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D015533:Transcriptional Activation; D014822:Vitreous Body
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D016650:Fluorescein-5-isothiocyanate