AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter.
OBJECTIVE
Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones.


METHODS
Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter.


RESULTS
The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not.


CONCLUSIONS
The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.
Khani
Shahrokh C
SC
Department of Ophthalmology, Ross Eye Institute, State University of New York, Buffalo, New York 14215, USA. skhani@buffalo.edu
Pawlyk
Basil S
BS
Bulgakov
Oleg V
OV
Kasperek
Eileen
E
Young
Joyce E
JE
Adamian
Michael
M
Sun
Xun
X
Smith
Alexander J
AJ
Ali
Robin R
RR
Li
Tiansen
T
D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D007668:Kidney / Q000196:embryology; D049408:Luminescent Agents; D051379:Mice; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D019572:Retinal Neoplasms / Q000235:genetics; D012175:Retinoblastoma / Q000235:genetics; D017299:Rod Opsins / Q000235:genetics; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D049408:Luminescent Agents; D017299:Rod Opsins; D049452:Green Fluorescent Proteins; D051606:G-Protein-Coupled Receptor Kinase 1