In vitro Analysis of Ribozyme-mediated Knockdown of an ADRP Associated Rhodopsin Mutation

Retinitis Pigmentosa (RP) is a group of retinal degenerative diseases that are characterized mainly by the loss of rod photoreceptor cells. RP can be sub divided into 3 classes, autosomal dominant, autosomal recessive, and X-linked RP, where the mutant gene exists on the sex chromosome (X) (Hartong et al., 2006; Wang et al., 2005). Mutations in rhodopsin are the most common cause of the autosomal-dominant form of RP (ADRP). More than 100 mutations in rhodopsin account for approximately 30% of ADRP cases with varying severity of visual impairment (Dryja et al., 1991). Based on in vitro studies (Sung et al., 1993; Sung et al., 1991), rhodopsin mutations that are similar to wild-type in terms of expression levels, folding and formation of functional photopigment are considered Class 1 mutations. On the other hand Class 2 mutants are those in which the opsin protein is inefficiently transported or is retained in the endoplasmic reticulum (ER). The mutation of Proline to Histidine at the 23rd amino acid of rhodopsin (P23H) is a Class 2 mutation. The Xenopus laevis in vivo model for P23H (Tam and Moritz, 2006) shows that the protein is misfolded and that is accumulated in the inner segment (IS) causing ER overload and cellular stress thus supporting its categorization as Class 2. It has been shown that the P23H substitution reduces the binding capacity for 11-cis retinal and causes a defect in glycosylation (Noorwez et al., 2004). The first symptom of ADRP associated with the P23H mutation is impaired night vision that appears between 10 to 30 years of age. The majority of these individuals became blind by the age of 60. Gene therapy is one of the most promising treatments for inherited retinal diseases. At the beginning it was a challenge to achieve successful gene therapy in the retina due to the lack of cell specific and efficient targeting as well as the lack of a delivery method that would cause minimum damage to the eye. Additionally, there were no specific animal models that closely mimicked the human disease presentations. So far, two types of approaches have been considered for gene therapy in ADRP animal models. The first is delivery of genes for neurotrophic factors such as GDNF (McGee Sanftner et al., 2001) and antiapoptotic proteins such as XIAP (Petrin et al., 2003) to prevent the ongoing death of the photoreceptors. The second approach is to block the synthesis of mutant proteins and increasing the ratio of wild-type vs mutant rhodopsin to delay the onset of retinal degeneration (Drenser et al., 1998; LaVail et al., 2000). For instance, Lewin and his colleagues used AAV2 to deliver ribozymes that cleaved the P23H rhodopsin mRNA and showed an effective treatment in a rat model of ADRP (LaVail et al., 2000). Recently various reports demonstrate the robust efficacy of RNA interference (RNAi) in animal models of human disease and support the RNAi based new therapeutic approach as one with the potential to change the treatment of human disease (Bumcrot et al., 2006). RNAi has been used to silence P23H human rhodopsin alleles by 94.9% in human embryonic retinoblasts (Cashman et al., 2005) thus providing further evidence that this approach is a viable one to treat specific human disease causing mutations.