Abstract
The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase -1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis – 1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies.
Highlights
Development of effective, long-lasting therapies for the treatment of progressive autosomal recessive retinal diseases that cause blindness early in life remains a challenge
The gene mutated in Leber congenital amaurosis – 2 (LCA2) encodes retinal pigment epitheliumspecific protein 65-kDa (RPE65), a protein that is expressed in pigment epithelial cells and is critical for processing the vitamin A chromophore that photoreceptors need to regenerate their visual pigments following light stimulation [8,9]
We found that treatment of the retinas of these animals with a lentivirus carrying a normal copy of the guanylate cyclase -1 (GC1) gene restored function to the infected photoreceptor cells, as evidenced by measureable electroretinograms and visual behaviors including the optokinetic nystagmus (OKN) reflex [15]
Summary
Development of effective, long-lasting therapies for the treatment of progressive autosomal recessive retinal diseases that cause blindness early in life remains a challenge. There have been numerous studies showing that the effects of these mutant genes on photoreceptor cells can be reversed by delivering a normal copy of the mutated gene to these cells; in most cases these corrective gene therapies only provide a temporary reprieve from photoreceptor degeneration and the ensuing blindness that defines these diseases [1,2,3]. Because many of these aggressive photoreceptor diseases cause blindness early in life, it is desirable to develop treatment strategies that provide lifelong therapeutic benefits. In addition to amplifying the effect of RPE65 therapy, the relationship between the pigment epithelium and the adjacent photoreceptors serves to minimize the number of untreated photoreceptor cells within treated areas which could positively influence the efficacy of the treatment if degeneration of untreated cells compromises survival of treated cells
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