Abstract
Abstract: :
Purpose: Mutation in the RPE65 gene results in translation of a truncated RPE65 protein leading to congenital retinal dystrophy in the RPE65 null mutation dog, a model for Leber Congenital Amaurosis (LCA). RPE65 is a 61kDa membrane associated protein preferentially expressed in the retinal pigmented epithelium (RPE). Gene therapy using a recombinant adeno associated virus (rAAV) has proven effective in restoring vision in affected dogs (Narfström K. et al., IOVS 2003), but treatment resulted in an inflammatory response (uveitis) in 9 of 12 animals. Our goal was to determine if treated dogs had circulating antibodies specific for the RPE65 protein up to 2 years following gene therapy. Methods:Canine RPE65 cDNA was cloned into a pcDNA3.1D/V5– His–TOPO vector (Invitrogen). The sequence of interest was amplified by PCR using a primer containing the IL–4 leader and RPE65 sequences. The plasmid was used to transfect the COS–7 cell line. The transfection supernatant was purified using a Ni–NTA column (Invitrogen). The recombinant protein was used as the coating antigen for indirect ELISA. Serum and aqueous humor from treated dogs and untreated controls were obtained at regular time points following surgery (2–48 mo.) and used as test samples. Results: We constructed a plasmid (pMLC. IL4–RPE65) that expressed the canine RPE65 protein containing a N– terminal IL–4 leader sequence and a C–terminal Histidine tag. The recombinant protein was confirmed by Western blot and silver stain. Results from the ELISA indicated that gene therapy treated dogs did not have higher systemic or ocular reactive titers specific for RPE65 than untreated dogs from the same colony. Conclusions: Expression and purification of the membrane associated protein, RPE65, was achieved by utilizing the leader sequence of a secreted protein (IL–4), using the engineered vector. The lack of RPE65 specific antibodies in the ELISA 2–48 mo. post–operatively indicated that the inflammatory response observed in some of the eyes following the intraocular injections were not due to humoral response against the RPE65 protein. This shows that long–term expression of the transgene can be achieved in the RPE without significant adverse immune reactions.
Keywords: gene transfer/gene therapy • retinal degenerations: cell biology • inflammation