Abstract
C
Choroideremia is often diagnosed by the unique appearance caused by progressive loss of retina, choroid, and retinal pigment epithelium (RPE). Men and boys are affected with this condition because of its X-linked inheritance, but some female carriers can be mildly symptomatic (Coussa et al., 2012), presumably depending on the degree of X chromosome inactivation. In contrast to Leber's congenital amaurosis (LCA), the first retinal degeneration target of gene therapy, choroideremia progresses slowly with a peripheral to central direction of retinal degeneration. In childhood, the main symptom of CHM is nyctalopia, or difficulty seeing in dim light. As exemplified by the patients in the current study, individuals in their 30s and 40s may only have small islands of foveal and macular retina remaining. The fovea is the central macular region of high visual discrimination (visual acuity and color vision). Thus, individuals with advanced choroideremia may have “20/20” visual acuity (i.e., can read an eye chart perfectly) but be unaware of obstacles in their path because of poor side vision caused by degeneration of the peripheral retina. Eventually even this tiny central window on the world is destroyed by the disease, which in severe cases becomes debilitating just as young men are completing their schooling and starting their careers and families. The awareness of progressively diminishing visual abilities can be psychologically crippling.
Clearly there is a great need for a treatment for choroideremia, but this disease also has some distinct advantages with respect to gene therapy: (1) The 1.9 kb CHM cDNA, which encodes Rab escort protein-1 (REP1), fits within the small cargo confines of the recombinant virus adeno-associated virus (AAV), a vector that has a strong safety record; (2) the disease results from functional null mutations in CHM, and gene augmentation therapy is thus an appropriate strategy; (3) the normal expression pattern of CHM has been characterized, and the affected cells (primarily photoreceptors and retinal pigment epithelium, RPE) can be targeted safely, efficiently, and stably with AAV2; (4) there is a quantitative assay that can be used to verify normal function conferred by the AAV.CHM; (5) proof-of-concept of gene augmentation therapy for choroideremia has been established in vitro and in vivo (Anand et al., 2003; Tolmachova et al., 2012, 2013; Vasireddy et al., 2013); (6) conditions whereby accurate dosing of AAV is assured had been developed (Bennicelli et al., 2008); and (7) the safety of subretinal and subfoveal injection of AAV serotype 2 (AAV2) and incorporation of the chicken beta actin constitutive promoter had been “de-risked” in longterm studies of LCA-RPE65 (Maguire et al., 2008, 2009; Bennett et al., 2012).
The present study also addresses unique challenges in developing gene therapy for choroideremia, including that (1) phase I safety studies in CHM, by necessity, enroll adults reliant upon limited areas of viable retina that typically include the fovea. As the thinnest part of the retina, the fovea is also the most vulnerable to mechanical injury, such as that inherent to subretinal injection of virus. (2) The cellular targets in CHM are both photoreceptors and RPE cells, whereas only RPE cells were the targets of LCA-RPE65. Thus, the target cell types are expanded in CHM compared to LCA-RPE65. (3) The primary goal in CHM gene therapy studies is halting progression of the disease rather than reversing blindness as it was in the LCA-RPE65 studies. Because CHM progresses slowly, the time point at which efficacy could be detected was uncertain.
The current report describes the 6-month results of unilateral subretinal delivery of AAV2.REP1 in six individuals with advanced CHM (Maclaren et al., 2014). The design of the vector/excipient was very similar to that used in the LCA-RPE65 study carried out at The Children's Hospital of Philadelphia (CHOP)(Bennicelli et al., 2008; Maguire et al., 2008) except for the transgene (CHM cDNA instead of RPE65 cDNA) and incorporation of the woodchuck hepatitis virus post-translational regulatory element. With the exception of an individual with foveal degeneration, the targeted area includes the fovea, as this was the only remaining viable tissue in these patients with advanced disease. Other investigators have voiced concern about carrying out subretinal injections in this region because of the vulnerability discussed earlier (Jacobson et al., 2012). To assure safe and controlled foveal detachment and vector injection, the investigators used a unique two-step approach rather than a single injection of AAV: the fovea was detached first using balanced salt solution (BSS), and AAV2.REP1 was then delivered to the area of detachment. The team presented the doses as dose/mm2 tissue (instead of the more usual dose/retina), although it is not clear whether dilution by the previously injected BSS was taken into account.
Although this study was run as a phase I safety trial, the results were encouraging from both safety and efficacy standpoints. There were no systemic safety concerns. Prior to the study, the predominant safety concern had been that the foveal detachment would result in reduced visual acuity. However, all patients recovered their baseline acuity and two of the patients showed large gains in acuity, with one patient gaining over three lines on the eye chart (the gold standard for significant improvement in low vision patients). In the one subject whose fovea was not exposed to vector, eccentric fixation was established on the region of surviving tissue that had been exposed to vector, suggesting that this region had become more sensitive because of the gene transfer. Moreover, all patients showed a trend of increased maximal light sensitivity measured with dark-adapted microperimetry with the degree of improvement correlating to the dose of vector relative to the area of viable tissue. In contrast, there was a decline in sensitivity in the untreated control eyes.
In summary, the short-term results from this small group of patients are very encouraging with respect to both safety and efficacy. Administration of AAV2 to the fovea caused no harm, and the patients tolerated the procedure well. Early indications even suggest that there may be efficacy. Since both the patients and the clinical team know which is the injected eye, and since the outcome measures all involve subjective tests (where the patient provides the answer), efficacy results must be evaluated with caution. However, because the design of this study allows comparison with an internal control (the untreated eye in this bilaterally symmetrical disease), it will be interesting to see over longer term follow-up whether the patients retain vision in the treated eye but not the untreated eye. A halting of disease would be a tremendous result. It will also be interesting, in future studies, to test the intervention in individuals with less advanced disease, when it may be easier to document further deterioration due to natural disease progression.