For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.

Background: The role played by microRNAs (miRs) as common regulators in physiologic processes such as development and various disease states was recently highlighted. Retinitis pigmentosa (RP) linked to RHO (which encodes rhodopsin) is the most frequent form of inherited retinal degeneration that leads to blindness, for which there are no current therapies. Little is known about the cellular mechanisms that connect mutations within RHO to eventual photoreceptor cell death by apoptosis. Results: Global miR expression profiling using miR microarray technology and quantitative real-time RT-PCR (qPCR) was performed in mouse retinas. RNA samples from retina of a mouse model of RP carrying a mutant Pro347Ser RHO transgene and from wild-type retina, brain and a whole-body representation (prepared by pooling total RNA from eight different mouse organs) exhibited notably different miR profiles. Expression of retina-specific and recently described retinal miRs was semi-quantitatively demonstrated in wild-type mouse retina. Alterations greater than twofold were found in the expression of nine miRs in Pro347Ser as compared with wild-type retina (P < 0.05). Expression of miR-1 and miR-133 decreased by more than 2.5-fold (P < 0.001), whereas expression of miR-96 and miR-183 increased by more than 3-fold (P < 0.001) in Pro347Ser retinas, as validated by qPCR. Potential retinal targets for these miRs were predicted in silico. Conclusion: This is the first miR microarray study to focus on evaluating altered miR expression in retinal disease. Additionally, novel retinal preference for miR-376a and miR-691 was identified. The results obtained contribute toward elucidating the function of miRs in normal and diseased retina. Modulation of expression of retinal miRs may represent a future therapeutic strategy for retinopathies such as RP.

The rhodopsin gene (RHO) encodes a highly expressed G protein-coupled receptor that is central to visual transduction in rod photoreceptors. A suite of recombinant 2/5 adeno-associated viral (AAV) RHO replacement vectors has been generated in an attempt to recapitulate endogenous rhodopsin levels from exogenously delivered AAV vectors in the retina of mice with a targeted disruption in the rhodopsin gene (Rho(-/-) mice). Approximately 40% of wild-type mouse rhodopsin mRNA levels (RNA taken from whole retinas) was achieved in vivo in AAV-RHO-injected eyes, representing approximately 50-fold increases in expression compared with the initial vector. The main focus of this study was to test whether expression of AAV-RHO replacement in Rho(-/-) mice provided therapeutic benefit, which to date had not been achieved. Rho(-/-) mice neither elaborate rod outer segments nor have rod-derived electroretinograms (ERGs). Our results indicate for the first time in this model that subretinal AAV-RHO delivery leads not only to RHO immunolabeling but the generation of rod outer segments as evaluated by light and transmission electron microscopy. Improved histology was accompanied by rod photoreceptor activity as assessed by ERG for at least 12 weeks postinjection. The most efficient AAV-RHO constructs presented in this study provide sufficient levels of RHO to be of therapeutic benefit in Rho(-/-) mice and therefore represent important steps toward generating potent AAV-RHO replacement genes for gene therapy in RHO-linked human retinopathies.

Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS‐peripherin‐linked autosomal‐dominant retinitis pigmentosa (RP), for which more than 50 disease‐related mutations have been identified to date. Mutation‐independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r‐rds, which has been altered to escape suppression but nevertheless encodes wild‐type protein) has been explored in COS‐7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r‐rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT‐PCR) and protein (ELISA) levels in COS‐7 cells. “Dual‐” and “triple‐expression” constructs carrying the shRNA suppressor and the marker EGFP with or without the r‐rds cassette were electroporated in vitro into retinal explants from 1‐day‐old pups. The retinae were dissociated at day 14, and transduced cells were FACS‐sorted using the coexpressed EGFP marker and analyzed by RT‐PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r‐rds was protected from suppression in COS‐7 cells. Similarly, efficient RNAi‐mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r‐rds was also achieved. These data validate the concept of RNAi‐based suppression coupled with replacement technology for the development of therapies targeting RDS‐linked autosomal‐dominant RP, and suggest that such approaches could potentially be used for other autosomal‐dominant diseases with similarly extensive intragenic heterogeneity. Hum Mutat 27(3), 260–268, 2006.

Recombinant adeno-associated virus (AAV) represents an efficient system for neuronal transduction. However, a potential drawback of AAV is its restricted packaging capacity of approximately 5 kb. To bypass this limitation, a number of dual- and triple-vector strategies divide the transgene(s) between two or three AAVs. The success of these approaches relies directly on efficient cotransduction of the component AAVs. Although proof of concept for these stratagems has been demonstrated, the underlying cotransduction rate has not been analyzed quantitatively. In this study, cotransduction efficiencies in both retina and hippocampus have been investigated, using two reporter AAVs expressing either a green (GFP) or red (DsR) fluorescent protein. Transduction efficiencies were monitored via microscopy, flow cytometry, and quantitative PCR. After viral transduction with 1.5×10 ⁹ viral particles of each of the reporter AAVs, approximately one-third of the retinal cells expressed one or both transgenes at levels detectable by native fluorescence. Notably, the majority of the remaining retinal cells were also transduced and expressed the reporters at lower levels, which were detectable only by immunolabeling. Flow cytometric analysis demonstrated cotransduction rates of up to 55% with the two reporter AAVs in retinal cells. Modifying the ratio of the two coadministered AAVs resulted in altered mRNA expression levels of the two reporter genes in cotransduced cell populations. The study suggests that codelivery of AAV is an efficient means of expanding the therapeutic application of AAV in neurons.