the treatment if degeneration of untreated cells compromises survival of treated cells. Unfortunately, unlike LCA2 therapies, the effects of corrective gene therapies designed to restore function to photoreceptors affected by genetic mutations located in photoreceptor genes are not amplified by retinal physiology or structure. Thus, developing methods to maximize the number of photoreceptor cells that receive corrective gene therapy remains a priority in developing treatments for these diseases. In addition to increasing the numbers of treated photoreceptors, can we prolong survival of these cells? One method to accomplish this would be to pair delivery of the corrective gene with delivery of additional therapeutic proteins that would enhance survival of photorecep- tors. The results of our work on LCA1 have led us to consider this possible treatment route. LCA1 is caused by mutations in the GUCY2D gene (NC_000017.10) that encodes guanylate cyclase-1 (GC1), an enzyme that is expressed in photoreceptor cells [11]. GC1 plays a critical role in the ability of photoreceptor cells to recover from light stimulation [12] and its absence in humans [13] and in the GUCY1*B avian model of LCA1 [14] results in severely compromised vision or blindness at birth. We found that treatment of the retinas of these animals with a lentivirus carrying a normal copy of the GC1 gene restored function to the infected photoreceptor cells, as evidenced by measureable electroretino- grams and visual behaviors including the optokinetic nystagmus (OKN) reflex [15]. The benefits of this therapy, which were clearly detectable in young animals, slowed but did not halt photoreceptor degeneration. We concluded from these results that corrective GC1 gene therapy is sufficient to restore function to photorecep- tors lacking this enzyme and that, if we could prevent the treated photoreceptor population from degenerating, then we may be able to reach our current therapeutic goal of inducing lifelong sight in the GUCY1*B model of LCA1. Over the past few years, we have been working to develop lentiviral-based, gene delivery strategies that would allow us to carry out meaningful studies of the effects of combination gene therapies on GUCY1*B photoreceptor function and survival. Ideally, the relative amounts of the proteins generated from these vectors should be highly consistent among infected cells. Our studies thus far have revealed that strategies involving delivery of either a mixture of two lentiviruses [16] or of lentiviruses carrying bicistronic transgenes constructed using a polio internal ribosome entry site (IRES) [15] fail to meet our protein expression criterion. Retinas treated with mixtures of two viruses contained very few infected cells that co- expressed the proteins encoded by the viruses in the mixture, a pattern that would be predicted if cells infected by one virus became refractory to additional infection [17]. In retinas treated with viruses carrying bicistronic IRES transgenes, the levels of protein produced from the cistron downstream of the polio IRES were often undetected in infected cells, a protein expression pattern that has been observed by others [18,19]. We also examined the protein expression characteristics of lenitiviral transgenes carrying two internal promoters, each driving the expression of a single cistron [16,20]. In general, both of the proteins encoded by the cistrons were detected in infected cells, but the relative amounts of these proteins among infected cells were highly variable. In the present study, we sought to resolve the problems encountered with these approaches by 1) using two identical promoters in our dual-promoter lentivectors, 2) inserting an additional WPRE element between cistrons in our dual-promoter vectors, and 3) developing vectors carrying bicistronic transgenes containing the pTV1 viral 2A-like cleavage sequence. Viral 2A-like cleavage sequences, which were first identified in studies of the foot Lentiviral Delivery of Proteins Using 2A Peptides and mouth disease virus [21], are short peptide sequences that, when fused in frame between two cistrons, trigger co-translational ‘ribosomal skipping’, a process that produces equimolar quantities of the proteins encoded by the transcript. Inclusion of viral 2A-like cleavage sequences in viral transgenes has been shown to be an efficient approach for obtaining multiple, functional proteins from a single vector in vitro and in vivo [22–24]. Our results show that the protein expression characteristics of the 2A-containing vectors are superior to those obtained using the other approaches examined in this study in terms of protein level and the reproducibility of the protein expression pattern among infected cells. Importantly, we also show that GC1, delivered to GUCY1*B photoreceptors in the context of a 2A fusion peptide, is able to restore function to these cells as evidenced by the appearance of OKN reflexes in the treated animals. Based on these results, we conclude that 2A-like cleavage peptides, while not a panacea for all gene therapies, can, with proper optimization, be used to create bicistronic transgenes that reliably express both of their encoded proteins. Additionally, these peptides are small. For these reasons, they should prove to be useful in the context of many of the viral vectors currently being used in gene therapy applications. Results Design of Bicistronic Lentiviral Vectors Three different bicistronic transgenes, each encoding GFP and hemagglutinin (HA)-tagged mCherry (mCherH) fluorescent re- porter proteins, were cloned into our pFIN lentivector backbone [20]. The first bicistronic transgene carries two identical internal EF1 promoters (pFIN-EF1-GFP-EF1-mCherH-WPRE, Figure 1A), each driving expression of one of the fluorescent proteins. This transgene was constructed with a promoter that is ubiquitously expressed and exhibits relatively high activity. It has been suggested that use of the same promoter in constructing dual- promoter vectors could lead to inadvertent DNA recombination during either viral packaging or processing of the viral RNA in the infected cells [25]; however, we did not see evidence of these problems when testing dual-promoter vectors carrying identical photoreceptor-specific promoters [16]. Thus, we chose to examine the performance of the EF1 dual-promoter transgene, a construct that would be useful in applications requiring higher protein expression levels. The second bicistronic transgene was construct- ed by inserting a second Woodchuck hepatitis posttranscriptional regulatory element (WPRE) between the upstream cistron and the second EF1 promoter of the dual EF1 promoter vector (pFIN- EF1-GFP-WPRE-EF1-mCherH-WPRE, Figure 1B). This modifi- cation has been shown to improve expression of dual-promoter vectors carrying two identical promoters [26]. Finally, the third bicistronic transgene was constructed using the well-characterized porcine Teschovirus (pTV1) 2A-like cleavage peptide. This peptide sequence produces two proteins from a single transcript by inducing co-translational ‘‘ribosomal skipping’’ [27]. During the ‘‘cleavage’’ process, 18 of the 19 amino acids comprising the 2A peptide, save the C-terminus proline, remain fused to the upstream peptide. The C-terminus proline remains attached to the N-terminus of the downstream peptide. This transgene was created by fusing the cDNAs encoding GFP and mCherH in frame with the pTV1(2A) sequence (pFIN-EF1-GFP-2A-mCherH- WPRE, Figure 1C). Expression of the Bicistronic Transgenes in Transiently Transfected HEK 293FT Cell Cultures In this series of experiments, we examined the expression characteristics of the three bicistronic transgenes in transiently PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20553