Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

Abstract

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.

Gene-based therapies for dominantly inherited retinopathies

“…Furthermore, significant histological and electrophysical benefit has been demonstrated in the P347S mouse model of RHOadRP by combining suppression and replacement in a single administration of two AAV2/5 vectors, one with an RNAi suppressor and the other providing a codon-modified RNAi-resistant RHO replacement gene ( Figure 2). 13 At 20 weeks post-injection, some control eyes gave vestigial rod-isolated electroretinography responses and the outer nuclear layer had almost completely disappeared. In contrast, 3-4 rows of photoreceptor nuclei remained in the outer nuclear layer of eyes treated with the dual component therapy and rod-isolated electroretinography responses of approximately 60 mV were recorded.…”

“…The approach involves the use of two components, suppression of both mutant and wild-type RHO alleles and provision of a suppression-resistant replacement RHO gene, engineered using codonredundancy. [7][8][9][10][11][12][13][14] In the context of dominant retinopathies, suppression and replacement is also being explored as a therapy for peripherin/ retinal degeneration slow-linked adRP, the next most common form of adRP after RHO-adRP. 15,16 In principle, replacement genes could be modified in a number of ways, for example, at degenerate sites as stated above.…”

“…Bars¼500 and 100 nm (inserts). Provided with permission from Millington-Ward et al 13 and Huntingtin involved in Machado-Joseph disease and Huntington's disease, respectively. 42 Indeed, before the exploration of suppression and replacement therapies, the strategy that was considered for dominantly inherited disorders typically involved selective suppression of the mutant allele.…”

“…However, benefit following gene delivery of a RHO gene was not observed in a different murine model for RHO-adRP, the P347S mouse. 13 In contrast to the scenario outlined above, there may be some dominantly inherited disease pathologies where the wild-type protein is not required for the survival and functionality of fully differentiated mature neurons, such as photoreceptors. Indeed, for such disorders suppression alone may provide a potential therapeutic strategy, that is, suppression of both wild-type and mutant alleles, representing a mutation-independent therapy.…”

Gene Therapy of Inherited Retinopathies: A Long and Successful Road from Viral Vectors to Patients

“…This is an interesting approach, as promoter repression targets the locus of a gene that is present in two copies per diploid cell, as opposed to the transcript, which can be very abundant and challenging to silence, as in the case of RHO. To date, the most advanced proof of concept of the ''suppression-replacement'' strategy for dominant RHO mutations has been provided by O'Reilly et al (2008), Mao et al (2012), andMillington-Ward et al (2011), who used a single AAV2/8 vector that codelivers an shRNA directed to RHO and a silencing-resistant RHO transgene. This strategy resulted in significant delay of PR cell loss in treated RHO-P23H mice (O'Reilly et al, 2007;Mao et al, 2012) and in increased rod function (Mao et al, 2012).…”