Gene Augmentation for adRP Mutations in RHO

As many as 40% of retinitis pigmentosa cases display autosomal dominant inheritance, and approximately one quarter of these are attributable to mutations in RHO, the gene for rod cell opsin (Sullivan et al. 2006). A complicating feature of RHO-based autosomal dominant retinitis pigmentosa (adRP) is its alleleic diversity: More than 100 disease-causing mutations have been mapped to RHO, and the vast majority are dominant (see https://sph.uth.edu/retnet/home.htm). These mutations can be classified both by their clinical presentation and by their impact on the cellular distribution of rhodopsin. Such distinctions are critical because they determine how, or even whether, a particular patient should be considered for gene therapy. Based on clinical observations, Cideciyan and coworkers (1998) described two classes of adRP. Class A patients lost retinal function over the entire retina and reported the onset of night blindness early in life. In contrast, class B patients exhibited a milder phenotype, normal rod activation kinetics, and preserved rod outer segment length, with anomalies in the rod visual cycle that were mutation specific. Photoreceptor degeneration in subclass B1 was not homogenous, and some patients showed an inferior to superior progression of disease. These authors conclude that the “catastrophic loss of rod function” in class A patients may not be correctable, so that therapy should be focused on preserving cones; whereas, in class B patients, rods have the potential to be rescued, and preserving rods is the best hope for protecting cones. The question of why cones die in retinitis pigmentosa is also crucial, because the answer will determine how many rods must be rescued to preserve central vision. Two classes of opsin mutations were also described as a result of studies in tissue culture cells (Sung et al. 1991; Kaushal and Khorana 1994). Class I opsin mutants were expressed at high levels in cells, reconstituted rhodopsin by binding 11-cis-retinal and partitioned to the plasma membrane. Class II mutants were expressed at low levels, did not bind 11-cis-retinal and remained in the endoplasmic reticulum. The relationship between the cellular behavior of mutant opsin proteins and the clinical impact of opsin mutations is not always clear, but a few generalizations can be made. Most of the mutations that lead to opsin misfolding and instability (i.e., class II) lead to a milder disease phenotype (class B). In contrast, mutations affecting the carboxyl-terminus of opsin, such as V345L and P347S, are expressed at high levels. These mutated proteins form rhodopsin, but lead to early onset, severe retinal degeneration (class A). Transgenic animal models of adRP have provided some clarification and some confusion. For example, rhodopsin with carboxyl-terminal chain terminating mutations (Q344X and S334X) is mislocalized to the plasma membrane of the inner segment (Sung et al. 1994; Li et al. 1996; Green et al. 2000), and this improper membrane insertion is toxic to rod photoreceptors. In contrast, class II mutations (P23H and T17M), which cause opsin to be retained in the endoplasmic reticulum, may cause cell death by continued activation of the unfolded protein response (Lin et al. 2007; Gorbatyuk et al. 2010; Kunte et al. 2012). Mendes and colleagues (2005) have refined the classification of RHO mutants, synthesizing results obtained in cell and transgenic models of adRP. One risk of transgenic models is related to the potential for overexpression of rhodopsin, which in itself may be toxic (Olsson et al. 1992a; Tan et al. 2001). Sakami and coworkers described a knock in mouse model of P23H Rho (the mouse version of RHO). Unlike transgenic P23H models, this line displayed the slow retinal degeneration characteristic of type B patients, and opsin did not accumulate in the endoplasmic reticulum but led to the disruption of disc membranes in rod outer segments (Sakami et al. 2011). Two critical questions must be answered when considering rod-directed gene therapy: (1) Is the degeneration progressing too quickly for rescue by gene delivery to rod photoreceptors? If rod cells have begun their cell death program when the diagnosis is made, then therapy must be directed at macular cones (Yang et al. 2009; Punzo et al. 2009; Usui et al. 2009; Murakami et al. 2012), or alternative strategies such as stem cell therapy, retinal prosthesis or gene delivery of light sensitive channels to cells of the inner retina may be indicated. (2) How does mutant opsin kill the cell? RHO mutations may be dominant for either of two reasons (Wilson and Wensel 2003; Mendes et al. 2005). Rhodopsin forms dimeric complexes in the disc membrane (Fotiadis et al. 2003), and mutant proteins might interfere with the function of normal rhodopsin or its assembly in the membrane, thereby exerting dominant negative effects. Alternatively, gain-of-function mutations could cause rhodopsin to be intrinsically damaging to the rod cell. It may be possible to treat dominant negative mutations by increasing the level of the normal protein (supplementation). For mutations that cause rhodopsin to be injurious, however, suppressing the expression of the mutant proteins may also be required.