## 4 Genome Editing for Autosomal Dominant Retinitis Pigmentosa
### Rho  
The _Rho_ gene encodes rhodopsin, a light-sensitive protein, which is involved in visual transduction. Rhodopsin is a G-coupled receptor, which comprises almost 50% of the total protein content of rod outer segments and 80% of that of discs [13; 57]. _RHO_ is the most common gene causing adRP, with the percentage of cases varying depending on the geographic area, up to 30% in the US [55; 58] and between 16%-20% in Europe [56; 59]. According to the Human Gene Mutation Database ([[http://www.hgmd.cf.ac.uk/ac/index.php](http://www.hgmd.cf.ac.uk/ac/index.php)]([http://www.hgmd.cf.ac.uk/ac/index.php](http://www.hgmd.cf.ac.uk/ac/index.php))), almost 220 _RHO_ mutations have been identified to cause different forms of RP. The adRP-causing mutations disturb protein structure and diverse functions, including folding, trafficking, endocytosis, membrane homeostasis, and proteasome degradation, and are grouped into classes depending on the function that is affected [54]. The majority of _RHO_ mutations that cause adRP are gain-of-function mutations [15]. A good example is the p.Pro23 His (P23H) mutation, which is the most common _RHO_ mutation associated with adRP. P23H belongs to the so-called class II RHO mutants, which do not fold properly [15; 54]. Due to their improper folding, class II mutants are labeled with ubiquitin and are destined for degradation by the ubiquitin proteasome system (UPS) [60]. Because of the large protein load, the degradation machinery is overwhelmed, which results in a failure to clear other misfolded proteins and leads to cell toxicity. Furthermore, in some cases, wild type rhodopsin becomes trapped with the class II mutant protein in the endoplasmic reticulum [22]. As a result, the wild type protein is more highly cleared by the proteasome [61] and is not delivered to the outer disc membrane [61; 62]. As such, we could say that class II mutants have a secondary dominant-negative effect. Given the high prevalence of _RHO_ mutations, and in particular P23H, it is not surprising that this gene has attracted the highest interest for gene therapy studies. As P23H partially exerts a dominant-negative effect, a simple strategy of supplementation with wild type rhodopsin has been studied. [PERSON] et al. provided wild type _RHO_ to P23H transgenic mouse using an AAV2/5 vector and showed reduced retinal degeneration up to 6-months post-treatment compared to non-treated controls [20]. However, one of the challenges of supplementation is that rods are highly sensitive to the levels of rhodopsin. [PERSON] and colleagues supplemented mice carrying the P23H allele with increasing numbers of wild type gene copies [21]. Although the authors confirmed that the dominant-negative effect of the mutant protein could be largely overcome by increasing the levels of the wild type protein, they reported that above three copies of the transgene, retinal deterioration persists; most likely due to a secondary effect of cell crowding. Thus, too low or too high levels of exogenous rhodopsin can lead to cell toxicity and retinal degeneration [21; 63]. Consequently, more emphasis has been given to supplementing exogenous rhodopsin while silencing the endogenous gene. Several groups have studied this approach by RNA interference technology. One group used AAV 2/5 vectors to deliver siRNAs that targeted the endogenous P23H _Rho_ allele while providing exogenous wild type _RHO_ to a heterozygous _Rho_*/- mouse model. The authors demonstrated improvement in ONL thickness up to 9 months post-injection [64]. Similarly, another group designed a study on a mouse model carrying another _Rho_ mutation, the p.Pro347 Ser (P347S) allele [65]. The authors used two AAV2/5 vectors: one to deliver the shRNA targeting the mutant allele and the second to deliver an extra copy of wild type _RHO_. Although an initial improvement was observed, the beneficial effect faded by 20 weeks post-injection.