Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Müller cells.
OBJECTIVE
Growing evidence suggests that successful treatment of many inherited photoreceptor diseases will require multi-protein therapies that not only correct the genetic defects linked to these diseases but also slow or halt the related degenerative phenotypes. To be effective, it is likely that therapeutic protein expression will need to be targeted to specific cell types. The purpose of this study was to develop dual-promoter lentiviral vectors that target expression of two proteins to retinal cones and rods, rods only, or Müller cells.


METHODS
Dual-promoter lentivectors were constructed using the following promoters: Xenopus opsin promoter (XOPS)1.3, murine opsin promoter (MOPS), interphotoreceptor retinoid binding protein promoter (IRBP156), rhodopsin kinase (RK), neural retina leucine zipper (NRLL), vimentin (VIM), cluster differentiation (CD44), and glial fibrillary acidic protein (GFAP). Vectors were packaged and injected into the neural tubes of chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter were analyzed in transduced, fully-developed retinas, using direct fluorescent and immunofluorescent microscopy.


RESULTS
IRBP156, NRLL, and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Müller cells; both VIM and CD44 were active in Müller and neural cells. Dual-promoter vectors carrying IRBP156 and RK or XOPS1.3 and MOPS, in the order listed, exhibited robust expression of both reporter transgenes in cones and rods or rods only, respectively. Expression of the upstream transgene was much lower than the downstream transgene in dual-promoter vectors constructed using two copies of either RK or IRBP156. Analyses of the expression of a dual-promoter vector carrying CD44 and VIM in the order listed showed that the activity of the VIM promoter was more restricted to glial cells when paired with the CD44 promoter, while the activity of the CD44 promoter was inhibited to the extent that no CD44-driven reporter protein was detected in transduced cells.


CONCLUSIONS
We have identified two dual-promoter vectors, one that targets cones and rods and one that targets rods alone. Both vectors reliably express the two proteins encoded by the transgenes they carry. When two well matched promoters are not available, we found that it is possible to target expression of two proteins to single cells using dual-promoter vectors carrying two copies of the same promoter. These vectors should be useful in studies of retina when co-delivery of a reporter protein with an experimental protein is desired or when expression of two exogenous proteins in targeted cells is required.
Department of Neuroscience, University of Florida McKnight Brain Institute, 100 Newell Dr., Rm L1-100 Box 100244, Gainesville, FL 32610-0244, USA. rowland@mbi.ufl.edu
Susan L
SL
Semple-Rowland
William E
WE
Coggin
Mero
M
Geesey
Kristofer S
KS
Eccles
Leah
L
Abraham
Krunal
K
Pachigar
Rachel
R
Ludlow
Shahrokh C
SC
Khani
W Clay
WC
Smith
D000818:Animals; D002642:Chick Embryo; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D051379:Mice / Q000235:genetics; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012160:Retina / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D019076:Transgenes; D014981:Xenopus / Q000235:genetics