Targeted transgene expression in muller glia of normal and diseased retinas using lentiviral vectors.
OBJECTIVE
Müller glia play crucial roles in retinal homeostasis and function. Genetic modification of Müller cells by viral gene delivery would be valuable for studies of their normal physiology and roles in retinal disease states. However, stable and efficient transgene expression in Müller cells after delivery of gene transfer vectors has remained elusive. Transcriptional and transductional targeting approaches were used to engineer recombinant HIV-1-based lentiviral (LV) vectors capable of highly efficient and sustained Müller cell transgene expression in healthy and diseased rodent retinas.


METHODS
Expression cassettes containing glia-specific promoters (CD44, glial fibrillary acidic protein, and vimentin) and an enhanced green fluorescent protein (eGFP) cDNA were cloned into LV backbones, which were packaged into infectious vector particles displaying either the vesicular stomatitis virus (VSV) or Ross River virus (RRV) envelope surface glycoproteins. Vectors were injected by intravitreal and subretinal approaches in wild type Sprague-Dawley (SD) and retinal degenerate S334Ter(+/-) transgenic rats aged 1 to 180 days. In vivo fluorescent fundus imaging and immunofluorescent confocal microscopy were used for comparison of expression efficiency, cell type specificity, and temporal expression characteristics.


RESULTS
The choice of viral pseudotype, regulatory promoter, and surgical delivery site each had a measurable effect on the level of eGFP transgene expression in Müller cells. The highest expression levels in SD retinas were attained with subretinal injection of VSV-G pseudotyped LV vectors containing the CD44 promoter. With these vectors, persistent eGFP expression in Müller glia was observed for more than 6 months, covering 25% to 30% of the retinal surface area after a single subretinal injection. Immunohistochemistry (alpha-glutamine synthetase) revealed that approximately 95% of the Müller cells were transduced in the region near the injection site. Delivery of these viral vectors and subsequent Müller cell eGFP expression had no negative impact on visual function, as assessed by electroretinography (ERG).


CONCLUSIONS
Pseudotyped LV vectors containing glia-specific promoters efficiently transduce and direct sustained transgene expression in retinal Müller glia. Vectors of this type will be useful for experimental treatment of retinal disease, as well as for physiological and developmental investigations of the retina.
Greenberg
Kenneth P
KP
Department of Vision Science, University of California-Berkeley, Berkeley, CA 94720, USA.
Geller
Scott F
SF
Schaffer
David V
DV
Flannery
John G
JG
D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D004596:Electroretinography; D005786:Gene Expression Regulation / Q000502:physiology*; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018960:Hyaluronan Receptors / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D018613:Microscopy, Confocal; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012162:Retinal Degeneration / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes; D014746:Vimentin / Q000235:genetics
D005904:Glial Fibrillary Acidic Protein; D018960:Hyaluronan Receptors; D014746:Vimentin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins