Total RNA was isolated from the RPE of a carrier Briard dog, by using extraction reagent (RNAzol B; Teltest, Friendswood, TX) and reverse transcribed with a kit (Retroscript; Ambion, Austin, TX). The normal RPE65 cDNA was amplified, by using oligonucleotide primers specific for canine RPE65 (forward 5′-TCC
CCG CGG CTC GAG ATG TCC ATC CAA GTG GAG CAT C-3′, and reverse 5′-CC
A TCG ATC
TCT AGA TTA GGA TTT TTT GAA CAG TCC-3′) and subcloned into a cloning vector (pBluescript; Stratagene, La Jolla, CA) using restriction sites included in the primers. This construct was sequenced by automated dideoxy sequencing on (model CEQ 2000; Beckman Coulter, Fullerton, CA), to verify the authenticity of the RPE65 clone. The verified RPE65 cDNA was then subcloned into the pCI vector (Promega, Madison, WI) with the human cytomegalovirus promoter (hCMV) on its 5′ end and the late SV40 polyadenylation signal (PolyA) on its 3′ end. The pCI vector was transiently transfected into Cos-7 cells. After 48 hours, cells were harvested and analyzed for expression of RPE65 by immunoblot analysis. The cassette containing the hCMV, RPE65 and PolyA was removed and subcloned into the plasmid AAV2 vector, pSSV9,
17 between the two inverted terminal repeats (ITRs). A control construct was made by replacing the RPE65 cDNA with the green fluorescent protein (GFP) cDNA. The resultant subclones, pAAV.CMV.RPE65 and pAAV.CMV.GFP were verified by restriction analysis. Cesium chloride gradient-purified pAAV.CMV.RPE65 and pAAV.CMV.RPE65 DNAs were transfected into human embryonic kidney 293 (HEK293 cells; ATCC, Manassas, VA). The pAAV.CMV.GFP-transfected cells were examined for GFP expression by fluorescence microscopy, the pAAV.CMV.RPE65-transfected cells were harvested at 48 hours after transfection, and expression of RPE65 was assessed by Western blot analysis. The excision and replication of the recombinant AAV (rAAV) DNA from the two constructs was verified by Hirt analysis.
18 The same batch of DNA was then used to produce rAAV constructs. Large-scale production of the rAAV constructs for use in the preclinical trials on dogs was performed by heparin-affinity chromatography at the Vector Core Facility of University of North Carolina Gene Therapy Center (Chapel Hill, NC), according to published methods.
19 No specific quality control studies were performed on the AAV constructs. The titers of the rAAV.CMV.GFP and rAAV.CMV.RPE65 were 2 × 10
10 transducing units (TU)/mL and 2 × 10
12 particles/mL, respectively. The rAAV viral constructs, resuspended in PBS, were stored at −70°C and thawed immediately before the subretinal injections.