## 4 Genome Editing for Autosomal Dominant Retinitis Pigmentosa
### Ppf531  
The pre-mRNA splicing factor, PRPF31, is a ubiquitously expressed component of the spliceosome. It is the second most prevalent gene to cause adRP, accounting for up to 10% of all cases [55; 56]. Although _PRPF31_ mutations are lethal in the homozygous state [70], it is still largely unknown why they only cause retinal disease in the heterozygous state. One suggestion is that photoreceptors are more sensitive to PRPF31 levels due to their high demand on the spliceosome machinery [71]. Along this line, photoreceptors have been shown to follow a special splicing program that produces high levels of alternatively spliced transcripts in comparison to other retinal cells [72].  
There have been 175 _PRPF31_ variants identified to date. Most of these are either large deletions or result in a premature termination codon whereby the mutant transcript is cleared through nonsense-mediated decay (NMD) [73; 74]. As large deletions are difficult to detect, the number of mutations may be underestimated. Mutant transcripts that are cleared by NMD are considered to be null alleles and the carriers are functional hemizygotes. As the onset of symptoms seems to correlate with protein levels, the mode of action of _PRP31_ mutations appears to be haplinsufficiency [73].  
A striking feature of _PRPF31_ mutations is the associated incomplete penetrance within the same family. It is believed that disease manifestation is linked to the levels of the wild type protein. Three different factors that may affect these levels have been proposed: The first is _CNOT3_, which acts as a negative regulator of _PRPF31_. Lower expression of _CNOT3_ leads to less inhibition of PRPF31 and no clinical signs of the disease. In turn, the amount of _CNOT3_ expression seems to be linked with an intronic polymorphism [75]. The second factor is MSR1, a minisatellite repeat element, which is adjacent to the _PRPF31_ core promoter [76]. When there is at least one higher expressing 4-copy repeat _MSR1_ allele, then the carriers of the _PRPF31_ mutation are asymptomatic. When the carriers only have a 3-copy repeat _MSR1_ allele, then they can be either symptomatic or asymptomatic (ibid). Lastly, two _trans_-acting expression quantitative trait locus (eQTL), one situated on a different chromosome and the other located near _PRPF31_ on the wild type allele, have also been proposed to modulate _PRPF31_ expression [77].  
Therefore, it seems that there are multiple mechanisms that regulate the expression of _PRPF31_, and that the amount of protein production is determinant for the development of the clinical signs. Consequently, there are theoretically two suitable approaches to treat the disease by gene therapy. The first is gene supplementation [78] but surprisingly, to our knowledge, there have been no such studies for _PRPF31_, even though the size of the open reading frame (1497 bp) is permissive for AAV-mediated gene transfer. However, a patent for such an approach has been filed ([[https://patents.google.com/patent/WO2016144892A1/en](https://patents.google.com/patent/WO2016144892A1/en)]([https://patents.google.com/patent/WO2016144892A1/en](https://patents.google.com/patent/WO2016144892A1/en))). A second approach would be to specifically correct the mutant allele by genome editing. Such an approach was used to target the pathogenic _PRPF31_ mutation, c.1115-1125 del11. This mutation was corrected in patient induced pluripotent stem cells (iPSC) using the CRISPR/Cas system and an ssODN as a template for HDR [79]. The corrected iPSC were then differentiated into photoreceptors and RPE, which showed improved cilia morphology and rescue of the phagocytic capacity, respectively, in comparison to untreated cells. Lastly, as there is not one prevalent _PRPF31_ mutation, an interesting alternative could be to target a modulator of _PRPF31_ (for example, to invalidate _CNOT3_), as this approach could potentially be applied to a larger subset of patients.