either a furin protease cleavage site or a furin site plus an amino acid spacer upstream of the GSG linker- 2A sequence [38,39]. To determine if addition of a furin site to our EF1-GFP-2A-mCher transgene could increase cleavage of the 2A polypeptide, we constructed and compared the cleavage efficiency of GFP-furin- 2A-mCher to that of GFP-2A-mCher. EF1-GFP-Furin-2A-mCher was constructed by inserting nucleotides encoding furin binding site (RAKR) upstream of the GSG-2A sequence. Western blot analyses of HEK 293FT cells expressing these transgenes revealed that addition of the furin site interfered with 2A cleavage (Figure S1). Very little of the polypeptide translated from the EF1-GFP- Furin-2A-mChe transgene was successfully cleaved compared to that translated from the EF1-GFP-2A-mCher transgene. It is possible that inclusion of an additional amino acid spacer between the furin site and the GSG linker-2A site would improve cleavage efficiency of our transgenes. We have not examined this possibility. Addition of a furin cleavage site upstream of the 2A peptide, the cleavage of which would effectively remove the 2A peptide sequence from the C-terminus of the protein encoded by the upstream cistron, has also been reported to improve the function of this protein [38]. To minimize disruption of function of the upstream protein we routinely place cistrons that encode proteins amenable to C-terminus modifications upstream of the 2A peptide sequence in our vectors. Our and other investigators experiences with 2A sequences clearly indicate that it is important to examine the performance of each 2A construct in the cellular context in which they will be used. In addition to the 2A transgenes examined in this study, we also examined the expression characteristics of bicistronic transgenes that were constructed using two independent EF1 promoters. Our decision to construct and examine the expression characteristics of the pFIN-EF1-GFP-EF1-mCher-WPRE vector in this study was prompted by the results of a previous study in which we found that the expression levels of two proteins encoded by dual-promoter vectors carrying two identical copies of a single photoreceptor- specific promoter, while not equal, were much higher than those obtained from IRES-containing transgenes [16]. The results of our analyses of the expression of pFIN-EF1-GFP-EF1-mCher-WPRE lentivirus in transduced HEK 293FT cells (Figure 2A–D) and in retinas (Figure 3A–F) were surprising because very little to no GFP expression was detected in infected cells. In retina, the GFP that was detected was not co-localized with mCher. Interestingly, inclusion of an additional WPRE element immediately after the upstream cistron (EF1-GFP-WPRE-EF1-mCher) did not signifi- cantly improve expression of GFP in transduced cells (Figure 2E– H) or retinas (Figure 3G–L). In fact, in our hands this modification, which was previously reported to enhance expres- sion of both cistrons in a dual-promoter lentivector carrying identical hSynapsin promoters [26], produced a lentivirus that expressed only one or the other cistron in the vast majority of the infected cells. This unique protein expression pattern appeared to be observed only when the vectors were delivered in viral form. Both reporter proteins were co-expressed in HEK 293FT cells transfected with either plasmid DNA. One possible explanation for this observation is suggested by the expression characteristics of two of our dual-promoter lentivectors. When the EF1 promoters (1446 bp) used in this study to construct the EF1-GFP-EF1- mCher-WPRE transgene are replaced with either two rhodopsin kinase (297 bp) or interphotoreceptor retinol binding protein (262 bp) promoters, the reporter proteins are co-expressed in the retinal photoreceptors infected by the lentiviruses [16]. While untested, it is possible that the size of the EF1 promoter, which is approximately 5 times larger than either of the photoreceptor promoters, increases the probability of deletion of one of the Lentiviral Delivery of Proteins Using 2A Peptides cistrons during either viral packaging or reverse transcription of the viral RNA genome in infected cells prior to integration via homologous recombination [40–42]. Although the results obtained from our EF1 dual-promoter lentiviruses were undesired in our experiments, exclusive expression of either cistron of a bicistronic vector in transduced cells may be useful in some experimental or therapeutic applications. One of the major focuses of our research program is to develop therapies for human LCA1. The animal model that we use in our studies is the GUCY1*B chicken, which is currently the only animal model for this disease that models both the genotype and phenotype of human LCA1. We have previously demonstrated that GC1 corrective gene therapy is sufficient to restore function to photoreceptor cells and reverse blindness in these animals [15], a finding corroborated by the results of this study. The primary difference between the lentiviral vectors used in these two studies was in the design of the bicistronic GC1 transgenes they carried. The bicistronic transgene used in our previous study contained an IRES element. In the current study, this element was replaced with a 2A-like cleavage sequence. Both bicistronic vectors produced sufficient amounts of GC1 to restore function to GUCY1*B photoreceptors and visual behavior to treated animals, but only the 2A transgene consistently expressed both of the encoded proteins. Importantly, our results indicate that the proline residue retained on the N-terminus of GC1 following co- translational ‘cleavage’ of the 2A peptide does not negatively affect the activity of the GC1 protein encoded by the GFP-2A- GC1 transgene. To date, we have not yet achieved permanent vision restoration in GUCY1*B animals using corrective gene therapy alone, a problem that plagues many investigators working to design therapies to treat aggressive inherited photoreceptor diseases caused by mutations in genes expressed in these cells [3,43,44]. In the current study, we noted that higher percentages of retinal transduction with our GC1 vector were positively correlated with the strength of the visual responses exhibited by the animal and the duration of the benefits of the treatment. Our successes using corrective GC1 therapy alone suggest that it might be possible to achieve life-long restoration of vision in our model system by simply increasing the number of photoreceptors expressing the GC1 transgene. The fraction of the photoreceptor cell population that must be treated to achieve this outcome is unknown, but it is likely that the spatial relationships of the treated cells to other treated and untreated cells in the retina will have an impact on this value. We are interested in determining this value since it would provide a useful benchmark for comparing the effectiveness of different treatment strategies. A second approach that may lead to life-long vision restoration in our model would be to combine delivery of GC1 to the photoreceptors with delivery of additional therapeutic molecules (e.g. neurotrophic or anti-apoptotic factors) to enhance survival of the photoreceptors. The results of this study show that we will be able to successfully deliver combination therapies to photoreceptors and other retinal cells using bicistronic transgenes carrying 2A-like cleavage sequences. Efforts are currently underway to determine if combination therapies can improve treatment outcomes in our model of LCA1. In summary, we examined the expression of three different bicistronic lentivectors in HEK 293FT cells and in neural retina and found that the lentivectors carrying transgenes containing 2A- like cleavage peptides were the only ones that reliably expressed both of the proteins encoded by the transgene and expressed them in a highly reproducible manner among infected cells. Since we were able to restore vision in GUCY1*B chickens using the 2A vector encoding GC1, our laboratory has now begun to develop PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20553