In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter.
BACKGROUND
Endothelial cells are a target for gene therapy because they are implicated in a number of vascular diseases. Recombinant baculovirus have emerged as novel gene delivery vectors. However, there is no information available concerning the use of endothelial-specific promoters in the context of the baculovirus genome. In the present study, we have generated a recombinant baculovirus containing the human flt-1 promoter (BacFLT-GFP) driving the expression of the green fluorescent protein. Transcriptional gene targeting was analyzed in vitro in different mammalian cell lines and in vivo in adult rat retinal vasculature.


RESULTS
BacFLT-GFP evoked the highest levels of expression in the endothelial cell line BUVEC-E6E7-1, similar to those reached by recombinant baculovirus carrying the CMV promoter (112% relative to BacCMV-GFP, n = 4). Interestingly, BacFLT-GFP directed high levels of expression in rat glioma C6 and in human glioblastoma CH235 cells (34.78% and 47.86% relative to BacCMV-GFP, respectively). Histone deacetylase inhibitors such as butyrate or trichostatin A enhanced the transcriptional activity of both BacCMV-GFP and BacFLT-GFP. Thus, in this study histone deacetylation appears to be a central mechanism for the silencing of baculovirus, independently of the promoter utilized. In vivo transcriptional targeting was demonstrated in adult rat retinal vasculature by intravitreal delivery of BacFLT-GFP and immunohistochemical staining with von Willebrand factor (vWF). Analysis by fluorescence microscopy and deconvolved three-dimensional confocal microscopy of retinal whole mounts obtained after 3 days of baculovirus injection showed that most GFP-expressing cells localized to the inner limiting membrane (ILM) and ganglion cell layer (GCL) and colocalize with vWF (70%, n = 10) in blood vessels, confirming the endothelial phenotype of the transduced cells.


CONCLUSIONS
Taken together, our results indicate that the restricted expression in endothelial cells mediated by the flt-1 promoter is not affected by the context of the baculovirus genome and demonstrate the potential of using recombinant baculovirus for transcriptional targeted gene expression into the eye vasculature.
Luz-Madrigal
Agustín
A
Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, México D,F, 04510, México. aluz@ifc.unam.mx
Clapp
Carmen
C
Aranda
Jorge
J
Vaca
Luis
L
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D051381:Rats; D011995:Recombination, Genetic; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics* / Q000378:metabolism
D049452:Green Fluorescent Proteins; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1