Bicistronic Lentiviruses Containing a Viral 2A Cleavage Sequence Reliably Co-Express Two Proteins and Restore Vision to an Animal Model of LCA1 Jonathan D. Verrier, Irina Madorsky, William E. Coggin, Mero Geesey, Michael Hochman, Elleanor Walling, Daniel Daroszewski, Kristofer S. Eccles, Rachel Ludlow, Susan L. Semple-Rowland* Department of Neuroscience, University of Florida McKnight Brain Institute, Gainesville, Florida, United States of America 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 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. Citation: Verrier JD, Madorsky I, Coggin WE, Geesey M, Hochman M, et al. (2011) Bicistronic Lentiviruses Containing a Viral 2A Cleavage Sequence Reliably Co- Express Two Proteins and Restore Vision to an Animal Model of LCA1. PLoS ONE 6(5): e20553. doi:10.1371/journal.pone.0020553 Editor: Amy C.Y. Lo, The University of Hong Kong, Hong Kong Received December 3, 2010; Accepted May 4, 2011; Published May 27, 2011 Copyright: 2011 Verrier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by NIH grants EY11388 (SSR) and an NIH Vision Core Grant EY08571 (http://www.nei.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rowland@mbi.ufl.edu Introduction Development of effective, long-lasting therapies for the treatment of progressive autosomal recessive retinal diseases that cause blindness early in life remains a challenge. Many of these diseases are caused by mutations in genes expressed exclusively in photoreceptor cells that disrupt their structure and function. 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; however, in most cases these corrective gene therapies only provide a temporary reprieve from photoreceptor degeneration and the ensuing blindness that defines these diseases [1–3]. Because many of these aggressive photoreceptor diseases cause blindness early in life, it is desirable to develop treatment strategies that provide lifelong therapeutic benefits. The most straightforward approach to achieving this treatment goal is to ensure that every photoreceptor in the diseased retina receives a copy of the corrective gene required to restore function to the cell before it has irreversibly committed itself to die. This strategy, while biologically sound, is currently unrealistic given the limitations of existing gene delivery methods. An alternate strategy to achieve this goal is suggested by examining the long-term therapeutic successes recently achieved using corrective gene therapy to treat Leber congenital amaurosis – 2 (LCA2) [4–7]. The gene mutated in LCA2 encodes retinal pigment epithelium- specific protein 65-kDa (RPE65), a protein that is specifically 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]. In the absence of this chromophore, photoreceptors are unable to respond to light and eventually degenerate [9]. In human retina, the ratio of retinal pigment epithelial cells to photoreceptor cells is approximately 1:22 [10], one pigment epithelial cell supporting the function of about 22 photoreceptors. Thus, for every retinal pigment epithelial cell treated, approximately 22 photoreceptor cells regain function, a relationship that essentially amplifies the therapeutic benefits of the RPE65 therapy. In addition to amplifying the effect of RPE65 therapy, the relationship between the pigment epithelium and the adjacent photoreceptors also serves to minimize the number of untreated photoreceptor cells within treated areas which could positively influence the efficacy of PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20553