Nonmotile or primary cilia are increasingly being appreciated as having a diverse array of important functions. The connecting cilium in photoreceptors, the link between the light-sensing outer segment and biosynthetic inner segment, occupies a position of central importance in photoreceptor cell biology. There are a number of retinal dystrophies and types of Usher syndrome that are caused by genetic defects affecting the connecting cilia. A partial list includes RPGR (X-linked RP), Lebercilin (LCA), and CEP290 (LCA). RPGRIP1 is the first ciliary protein to be addressed in a replacement gene therapy approach, and the results from our study could prove important for the design of gene therapy protocols for other forms of ciliary defects that lead to photoreceptor degeneration. The present study was designed to test the potential therapeutic efficacy of a human-derived RPGRIP1
replacement gene in a murine model of LCA lacking RPGRIP1. This study is a follow-up to our previous study in which we showed that a RPGRIP1
replacement gene of murine origin led to improvement in photoreceptor function and survival in RPGRIP1–/–
mice (Pawlyk et al.
). In the previous study, we used a promoter derived from the mouse rhodopsin gene that drove expression primarily in rod photoreceptor cells. As a result the rescue was limited to rod photoreceptors. For human gene therapy, it is imperative that the replacement gene be derived from the human genome, and, in the case of RPGRIP1
, its expression needs to target both rods and cones. We have established that a promoter derived from the human rhodopsin kinase gene drives transgene expression in both rods and cones (Young et al.
; Khani et al.
; Sun et al.
). The relatively small size of the RK promoter (~200 bp) is ideal for use in combination with the RPGRIP1
replacement gene because the latter is relatively large (~4
kb), approaching the packaging size limit of AAV vectors. This short RK promoter was incorporated into our replacement gene design to drive a human RPGRIP1
replacement gene. A further technical difference from the previous study is that we chose to package the therapeutic gene construct into AAV2/8 to take advantage of the known benefits of this version of AAV vector, such as faster onset of transgene expression and higher expression levels in photoreceptor cells (G.S. Yang et al.
; Allocca et al.
; Natkunarajah et al.
). The AAV2/8 vector proved successful in driving rapid expression because we could detect human RPGRIP1 protein by immunofluorescence as early as 1 week postinjection. These data represent a marked improvement over a prior unpublished study of similar design and scope, in which we packaged the replacement gene in an AAV2/5 vector. Human RPGRIP1 expression from the AAV2/5 vector in recipient mice was lower than what was found in the present study, and the therapeutic efficacy appeared minimal (our unpublished data).
The human RPGRIP1 protein expressed from the replacement gene was identical in apparent molecular weight to that of the native RPGRIP1 from human donor retinal tissues. The human RPGRIP1 is predicted to have a molecular mass of 147
kDa, and the major mouse RPGRIP1 variant is predicted to have a molecular mass of 152
kDa. These are considerably smaller than the values (170 and 190
kDa) that we estimated on the basis of motility in sodium dodecyl sulfate (SDS)–polyacrylamide gels. We believe these differences can be reasonably explained by the high content of negatively charged amino acid resides (glutamic acid) in these proteins, especially in mouse RPGRIP1. A higher acidic residue content is known to retard motility of the polypeptides on SDS–polyacrylamide gels, thus giving higher apparent molecular weight estimates (Graceffa et al.
; Korschen et al.
; Iakoucheva et al.
). For example, the RPGR protein exhibits a similar behavior because of high glutamic acid content, having a much higher apparent molecular weight than would be predicated from its sequence. We conclude that the human RPGRIP1 coding sequence used in the replacement gene construct is indeed the same form that is expressed endogenously in human photoreceptors.
The expression level of human RPGRIP1 protein in recipient mouse retinas appeared lower than that of endogenous RPGRIP1 from WT mouse retinas. The reason for the lower expression is not fully understood at this time. We do not believe this is related to the strength of the RK promoter because expression levels from the transgenes were all quite high in at least four other constructs using this promoter. One explanation is that the inner retinal cells from the WT and human retinas also contributed to the total RPGRIP1 content on immunoblots, which would result in an underestimate of the expression level in the treated retinas. This possibility can be clarified in future experiments. Other explanations may be a reduced stability of human RPGRIP1 transcript, a shorter half-life of human RPGRIP1 protein, or difficulty in human RPGRIP1 protein folding in a mouse photoreceptor milieu. If the last hypothesis is correct, one would expect that this same vector should yield a higher expression level in recipient human photoreceptors in a clinical application. Even if the transgene expression level remains less than the endogenous level, we do not believe that poses a significant barrier to early clinical studies. The RK promoter exhibited excellent tissue specificity; we have not observed ectopic expression in RPE, inner retina, or nonocular tissues. Nor have we observed any apparent pathology or increased mortality in mice that received vector injections.
Human RPGRIP1 protein is functional in mouse photoreceptors as demonstrated both by protein localization studies and by better photoreceptor function (ERG) and morphology after treatment. Differences were seen in both the quality and numbers of rods and cones between the control and treated eyes. There was an improvement in rhodopsin and cone opsin localization patterns in the treated retinas. Treatment significantly promoted photoreceptor survival and improved photoreceptor morphology in terms of inner/outer segment length and organization, which are important indicators of photoreceptor general health. Retinal function was also greatly improved by treatment. Treated eyes had significantly larger rod and cone ERG amplitudes at all time points tested, and the rate of ERG decline was markedly slowed. These data confirm a therapeutic efficacy for this replacement gene construct in mouse photoreceptors and establish a prototype design for future clinical application in LCA patients with RPGRIP1 deficiency.
Despite marked improvement in retinal function and morphology, rescue of the retinal disease phenotype was not complete. By comparison with WT mice, treated RPGRIP1–/–
mouse eyes had rod and cone ERG amplitudes that were on average only approximately one-third of WT mouse values at the end point of the study (5 months). It should be noted, however, that subretinal injections in mice can acutely reduce ERG amplitude by as much as 50% (our unpublished observation). Therefore damage associated with subretinal injection could, in part, contribute to the incomplete rescue in the treated eyes. On the other hand, the data show that retinal function continued to decline over time in the treated eyes, as if the treatment did not fully reconstitute RPGRIP1 function in the recipient retinas. We could think of two main reasons to account for this. First, the expression level of transgenic human RPGRIP1 is substantially lower than that of endogenous RPGRIP1 in mice. Second, there is substantial divergence between human and mouse RGPRIP1 sequences, with some regions of the protein bearing no homology between the two species. This is in sharp contrast to the typically high degree of conservation between the two species for many other photoreceptor-specific proteins (e.g., those involved in the phototransduction cascade). Either of these reasons could have accounted for the incomplete rescue observed in this study. Yet another possibility is the existence of RPGRIP1 variants (Castagnet et al.
; Lu and Ferreira, 2005
; Won et al.
). It remains unclear if and to what extent these variants are functionally significant in photoreceptors. The reported variants represent variant portions of the N-terminal RPGRIP1 and do not contain the RPGR-interacting domain located at the C terminus. Therefore, they are not expected to participate in the core function of RPGRIP1, that is, to anchor RPGR in the connecting cilia. Further studies may be necessary to examine the individual roles of the variants in photoreceptor maintenance and function. Given the available data, it is reasonable to predict that the human RPGRIP1
replacement gene may produce a more favorable outcome in the human photoreceptor environment than in the murine photoreceptor environment.
The results from this study could pave the way for a future clinical trial targeting LCA caused by RPGRIP1 functional deficiency. The rapid disease progression in LCA presents a significant challenge to gene therapy because retention of sufficient photoreceptors in the retina is a prerequisite for a satisfactory therapeutic outcome. In this regard, it is encouraging that LCA associated with RPGRIP1
gene mutations has been reported by some to present with a more stable and somewhat nonprogressive disease course after the initial rapid decline in visual function (Hanein et al.
). Photoreceptors in the central retina appear to persist for long periods of time after visual function becomes unmeasurable (Jacobson et al.
). In our own clinic, we have also found by optical coherence tomography that patients with LCA due to RPGRIP1
mutations can retain a substantial number of photoreceptors even when visual function has largely been lost as measured by visual field and ERGs (our unpublished observations). These clinical observations highlight the potential for treatment in patients who carry RPGRIP1