Lentiviral Delivery of Proteins Using 2A Peptides Figure 5. Analyses of the optokinetic (OKN) reflex responses of GUCY1*B chickens treated with pFIN-EF1-GFP-2A-GC1-WPRE virus. (A) Frame of OKN video showing the optokinetic testing apparatus. Videos of the OKN responses obtained from wild-type (Video S1), untreated GUCY1*B (Video S2) and treated GUCY1*B chickens (Video S3) are provided as supplemental data. (B) Summary of OKN responses obtained from seven treated GUCY1*B animals. The plotted values are the means6SEM of the average OKN responses obtained in response to either the large bar (LB) or small bar (SB) stimuli. (C, D) Whole mounts of the left (C) and right (D) retinas of a 5-week-old animal that had been treated with the virus. The percentage of the total retinal area infected by the virus was estimated using ImageJ software. The orientation of the retinas (D-dorsal; T-temporal) is indicated in panel C. doi:10.1371/journal.pone.0020553.g005 period. The estimated total area transduced in the retinas of animals whose OKN responses declined over the test period ranged from 3–7%. The strength and stability of these responses over time were very similar to those that we observed in animals treated with our IRES GC1 bicistronic vectors [15]. Overall, the results of the current study are encouraging because they show that the GC1 encoded by the 2A transgene is functional and that visual responses can be generated by a relatively small number of functioning photoreceptor cells. Importantly, they also suggest that relatively small increases in the total number of transduced photoreceptors in treated retinas could significantly increase the strength and stability of visual responses in treated animals. Discussion The results of this study clearly show that it is possible to obtain consistent expression of two proteins from viral transgenes consisting of two cistrons joined in frame by the pTV1 viral 2A- like cleavage sequence. Importantly, the results of our study establish the therapeutic potential of this specific vector design. We found that we were able to reverse blindness in GUCY1*B chickens by treating them with pFIN-EF1-GFP-2A-GC1-WPRE virus, a finding that indicates that the GC1 encoded by this transgene was properly processed and was expressed in sufficient amounts in photoreceptor cells expressing the transgene to support the function of these cells. Together, these results highlight the potential of this vector design to serve as a platform for development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies. The cleavage characteristics and small sizes of 2A-like cleavage sequences make them particularly useful in developing multi- cistronic viral vectors [22]. Since the proteins encoded by transgenes containing 2A sequences are translated from one transcript, co-translational cleavage of these polypeptides is expected to produce equimolar quantities of the proteins [32– 34]; however, proper cleavage and post-translational processing of the proteins does not always occur as expected. The cleavage efficiency of the polypeptide can be influenced by the nucleotide sequences flanking 2A and the order of the cistrons in the transgene relative to the 2A sequence [23,28,35–37]. The order of the cistrons relative to the 2A sequence can also alter post- translational processing and intracellular trafficking of the proteins encoded by the cistrons [28,36,37]. The small size of the 2A sequence is a second advantage of this technology, a feature that is particularly attractive when constructing multicistronic transgenes using viral vectors with limited cargo capacity. Two counter indications to using 2A peptides are poor cleavage efficiency of the translated polypeptide and disruption of function of the upstream protein by the residual 2A peptide that remains fused to its C-terminus. Poor cleavage efficiency could lead to accumulation of significant amounts of uncleaved protein and the formation of toxic protein aggregates in cells expressing the transgene. Strategies that have been reported to significantly improve cleavage efficiency of 2A polypeptides include insertion of PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20553