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G.H. Travis, W.M. Pardridge, S. Nusinowitz, R.A. Radu, A.B. Roller; Attempted Rescue of the Phenotype in rpe65–/– Mice by Intravenous Administration of Immunoliposomes Containing an Rpe65 Expression Construct . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4763.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Rpe65 is an abundant protein in the RPE that binds all–trans–retinyl esters (atRE's), which it presents as substrate to the isomerase. Mice with a knockout mutation in the rpe65 gene contain virtually no 11–cis–retinoids and accumulate atRE's. Mutations in the human RPE65 gene are responsible for a severe recessive blinding disease called Leber's congenital amaurosis. Successful gene therapy has been reported in rpe65–/– mice and dogs using recombinant AAV vectors. This treatment requires subretinal injections and frequently causes uveitis in the injected eye. In this study, we attempted an alternative strategy for gene therapy of rpe65–/– mice using pegylated immunoliposomes (PIL's) as a gene–delivery system. Methods: We prepared expression constructs containing the coding region for wild–type or L450M–substituted mouse Rpe65 under control of the SV40 early promoter and enhancer. These constructs were used to transfect HEK293 cells as a test for Rpe65 expression by immunoblotting. DNA from these constructs was then encapsulated into liposomes containing distearoylphosphatidylethanolamine conjugated to polyethyleneglycol. Following solvent evaporation, the lipid mixture was sonicated to yield multilamellar vesicles. These were extruded through polycarbonate filters to yield DNA–containing, pegylated liposomes of 75–100 nm. Thiolated monoclonal antibodies against the mouse transferrin receptor were coupled to the liposomes. These were injected intravenously into wild–type and rpe65–/– mice. Results: Similar PIL's containing a ß–galactosidase reporter plasmid were shown to be expressed at high levels thoughout the RPE 48 hours following a single intravenous injection into wild–type mice. In the current study, we assayed for Rpe65 expression in treated rpe65–/– mice by real–time PCR and immunoblotting. Subsequently, we measured levels of rhodopsin, 11–cis–retinoids, and atRE's in the retinas and RPE. We also looked for functional rescue of the rpe65–/– phenotype by electroretinography. Conclusions: PIL's offer important advantages over recombinant viruses as a gene–therapy delivery system for inherited defects in the RPE. These advantages include uniform, high–level expression in RPE cells, non–integration of the expression construct into the host genome, minimal immunogenicity, and no requirement for intraocular injection. A balancing disadvantage of PIL–mediated gene therapy is the requirement for repeated biweekly intravenous injections.
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