Purchase this article with an account.
K.P. Greenberg, S.F. Geller, D.V. Schaffer, J.G. Flannery; In vivo Targeting of Müller Cells in the Rodent Retina Using Novel Lentiviral Vectors . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5206.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:Efficient transgene delivery and stable expression in Müller cells will be a valuable tool for therapeutic and physiological investigations of the retina. Previous studies using lentiviral and adeno–associated viral (AAV) vectors have achieved only limited Müller cell transduction or poor specificity following intra–ocular injection. Methods: Recombinant HIV–1 vectors were pseudotyped with envelope glycoproteins derived from either the Ross River Virus (RRV) or Vesicular Stomatitis Virus (VSV). Vectors were packaged by transient transfection of 293T cells and high titer viral stocks were obtained after ultracentrifugation. A panel of pseudotyped vectors were constructed in this way that contain either Müller cell–specific (glial fibrillary acidic protein, vimentin, glutamine synthetase) or promiscuous (CMV, CMV–ß–actin, ubiquitin) promoters to drive GFP reporter gene expression. Virus titer was quantified in vitro on primary Müller or 293T cells using real time PCR and direct GFP visualization. Concentrated virus was injected either subretinally or intravitreally into anesthetized Sprague–Dawley and S334Ter heterozygous rats. GFP expression was assessed in vivo by several methods: fluorescence fundus imaging, confocal fluorescence microscopy of retinal flat mounts, cryosections, and thick agarose sections. Retinal sections were double labeled with Müller cell specific antibodies to correlate Müller cell structures with virus–mediated GFP expression. Results: RRV and VSV pseudotyped lentiviral vectors containing Müller cell specific promoters effectively directed transgene expression specifically to Müller cells in the rat retina. GFP reporter gene expression was colocalized with antibodies to known Müller cell markers. In contrast, vectors delivering promiscuous promoters directed GFP expression primarily to the RPE or photoreceptors. Conclusions: Lentiviral vectors can be specifically engineered to target Müller cells in vivo by engineering the transduction and transcriptional characteristics of the viral envelope and promoter. Studies are ongoing to maximize the efficiency of transgene delivery by lentiviral vectors and compare them to similarly engineered AAV and adenoviral (Ad) vectors in vivo. We anticipate that these vectors can be used to deliver secreted proteins to retinal cells without the inherent trauma of subretinal injection.
This PDF is available to Subscribers Only