What has been learned after nine RPE65
-LCA young adult patients, representing three different studies, underwent uniocular subretinal gene therapy and were monitored for at least 1.25 months (Maguire et al.
) and at most 1 year (Bainbridge et al.
) ()? There has been no evidence to date of systemic toxicity of the retinal injections of vector in any of the patients. The concern over immune responses in gene therapy trials is high (reviewed in Zaiss and Muruve, 2008
). All three RPE65
-LCA trials performed studies to identify humoral immune reactions to AAV2 and assayed by ELISpot for a T cell-mediated response. In each case no positive result was evident. In the ASR test for AAV2 capsid-stimulated lymphocyte proliferation, which only this study included, one patient at the 90-day time point exhibited a stimulation index that just exceeds one of the previously established levels of significance (Brantly et al.
) but not another (Hernandez et al.
). This value was only slightly higher than at baseline and its relevance is unclear at present. This patient's ASR stimulation index will be reassessed at follow-up (see Supplementary Table 2
)to understand better these results and their variation with time. Overall, however, the fact that little if any immune response was measurable in any patient may not be surprising given the small doses delivered subretinally in each of these studies relative to rAAV2 vector doses commonly administered in nonocular gene therapy trials; these are typically 2–4 orders of magnitude higher. In addition, the relative immune privilege of posterior ocular spaces is well appreciated (Streilein, 2003
) and may serve to further limit any potential immune response to subretinal rAAV2 vector.
A key question for future RPE65
LCA clinical trials concerns how the outcomes of the three current trials relate to the respective titers of vector delivered in each case. Unfortunately, there are at least four levels of uncertainty directly related to the vector in such an analysis that largely preclude any clear conclusions. First, each study made, purified, and titered their vector in a different facility using different protocols without reference to a common standard or to each other's vector. Thus, each stated titer cannot be related with confidence to the other two. Second, the area of RPE/neural retina exposed to vector within the injection bleb, necessary to estimate an effective “multiplicity of infection” for vector particles per RPE cell, is not identical, or even knowable, in all three studies. This trial and Maguire and coworkers (2008
) delivered a 150-μl volume, and might be compared (but see preceding and following information). In Bainbridge and coworkers (2008
ml of vector was delivered subretinally and the resultant bleb was then moved by gas manipulation over an unknown, and perhaps unknowable, retinal area in order to expose the fovea to vector in each patient. Such manipulation of the vector bleb after injection will in effect reduce the vector titer within any repositioned bleb by an unknown amount due to vector binding to RPE cells and photoreceptors in the initial detached area. In addition, as the initial bleb is relocated to its final position, vector binding to newly exposed RPE and photoreceptors will occur as the bleb transits between them. Thus the effective amount of vector in the bleb's final position is difficult to estimate. Third, each of the three vectors was constructed with different regulatory elements controlling expression of the human RPE65 cDNA. Maguire and coworkers (2008
) employed a CBA promoter, like the present trial, but inserted a modified Kozak translational initiation sequence before the RPE65 cDNA (Bennicelli et al.
), unlike the natural initiation sequence used in this trial. Although asserted to optimize transgene expression, side-by-side experiments with a similar vector containing the natural initiation sequence was not reported. Bainbridge and coworkers employed a short RPE65 promoter that, although targeting expression primarily to RPE cells, is of unknown transcriptional strength relative to the CBA promoter-driven expression in RPE cells. Thus, the relative expression strengths of each of the three vectors on a per-vector genome basis cannot be confidently assessed. Fourth, the large differences in visual function at baseline among the cohorts of patients in each trial, as discussed previously, might suggest that the relative availability of RPE cells versus photoreceptors to interact with vector in the subretinal vector bleb could be significantly different from cohort to cohort, particularly if the ratio of RPE cells to photoreceptors varies progressively with loss of visual function. Thus, it is not currently possible to reliably comment on how the amount of vector delivered relates to any differences in clinical outcome among the three trials.
Retinal complications did occur. These included a full-thickness macular hole in patient 2 of Maguire and coworkers (2008
) and foveal thinning defined by high resolution optical imaging in P1 of the current study. Both of these patients had vector injections into areas that included the fovea; and both retinotomy sites were within 1–1.5
mm of the foveal region (Maguire et al.
; ). Other subretinal injections with retinal detachments that included the fovea (n
4) had retinotomies at further eccentricities, near the superior retinal vessel arcade. No measurements of foveal thickness before and after these injections, however, were reported (Bainbridge et al.
; Maguire et al.
). Retinotomies near the fovea would appear (on current evidence) to be an ill-advised strategy that may lead to disruption of foveal structure. Although subretinal injection is intricate and complex microsurgery, the procedure has many currently uncontrolled variables that could affect outcomes. This includes the alignment of the fluid stream during vector injection from the small-gauge needles in relation to the fovea, the exact trajectory of the fluid stream, and the degree of frailty of foveal photoreceptors and RPE that are chronically stressed by degenerative retinopathy. Even in normal retinas, there can be incomplete recovery of foveal vision after successful anatomic repair of fovea-off retinal detachments (Burton, 1982
; Ross and Kozy, 1998
; Schocket et al.
). Subretinal injections in the proximity of the fovea are expected to cause the largest separation between the neural retina and the RPE, one of the factors related to visual acuity recovery after retinal detachment with foveal involvement (Ross et al.
). Epiretinal membrane contraction, a common feature of retinal degenerations (Milam et al.
), has been mentioned as a possible cause of the macular hole in patient 2 of Maguire and coworkers (2008
) but is unlikely to be the sole determinant of this complication (Moshfeghi et al.
Efficacy in these early safety studies, although a secondary outcome, is of high interest to the field of gene therapy and to those seeking treatment for otherwise incurable hereditary retinal degenerations (Bok, 2004
). The standard outcome measure in ocular studies is visual acuity (Ferris et al.
). High-resolution visual acuity is subserved by the fovea, the primate-specific central retinal region with the greatest density of cone photoreceptors and unique neural connectivity (Provis et al.
). There was no proven increase in foveal
visual acuity in any of the nine patients, despite the fovea being included in the subretinal injections in six of the nine patients. The only study to report improved visual acuity is that of Maguire and coworkers (2008
), but these three patients had the lowest visual acuities at baseline of all the studies and the improvements did not increase visual acuity even to the level of the baseline acuities in the other two studies. The fact that a macular hole resulted from surgery but failed to be visually significant confirms the conclusion that any positive change due to treatment was from extremely severe vision loss to less severe vision loss. At lower resolutions (typically worse than 20/200), visual acuity may also be subserved by extrafoveal retina. The Maguire and coworkers (2008
) result, although suggested to be a placebo effect due to the low level of vision being measured (Miller, 2008
), is more likely real and attributable to para- or perifoveal increases in visual sensitivity.
Another measure of efficacy, dark-adapted sensitivity, targets the characteristic visual deficit in RPE65
-LCA patients: the >4-log unit reduction in light sensitivity in this disease in humans and in animal models (Jacobson et al.
; Aguirre et al.
; Roman et al.
). All three patients in the Maguire and coworkers study (2008
) self-reported “improved vision in dimly lit environments” but dark-adapted visual measurements were not reported. Bainbridge and coworkers (2008
) performed dark-adapted perimetry in all three patients and found increased function only in their patient 3. In the present study, all patients also self-reported an increase in vision under dim light conditions and there was a statistically significant increase in measured dark-adapted sensitivity posttreatment compared with baselines. None of the studies to date have determined whether an increment in sensitivity was mediated by rod or cone photoreceptors or both. The visual cycle subserving rod and cone photoreceptors appears to differ (Travis et al.
) and it is uncertain how RPE65
gene replacement will differentially affect this chronically abnormal and key retinoid pathway in rods versus cones.
What is the next step for this clinical research? Conclusions from the previous two studies suggest that any lack of efficacy is due to the advanced stage of retinal disease at baseline and there is thus a need for both higher doses within the fovea and prompt advance to children with RPE65
-LCA (Bainbridge et al.
; Maguire et al.
). More recent work, however, has indicated that there is no strong relationship between age and retinal structure or function until after the fourth decade of life (Jacobson et al.
). The present study suggests a need for greater understanding of the positive and negative effects of subretinal injection on retinal structure and visual function. Items worthy of attention in the group of nine patients already treated include the following: identification of the precise retinal location of any positive treatment effects, measurement of the magnitude of the effect across the treated zone, determination of the rod and cone photoreceptor contributions to the observed treatment effects, establishment of the relationship between the magnitude of any effect on baseline measures of photoreceptor and RPE integrity, and full explanation of any treatment failures revealed by sensitive noninvasive testing in this era of sophisticated high-resolution ophthalmic imaging.