The gene therapy group had a trend towards

The gene therapy group had a trend towards fewer ranibizumab retreatments than the control group, and in a post hoc analysis 11 (53%) gene therapy patients received ≤2 ranibizumab retreatments, with the majority having improved vision. A cohort of the gene therapy group received >2 ranibizumab retreatments, but lost or gained <5 letters, suggestive of either under treatment or a bimodal response alluding to either lack of gene therapy effect, less susceptibility to anti-VEGF therapy, or greater intrinsic disease activity. We were not able to identify any reliable indicators to suggest a cause for this potential divergent response such as pre-existing immunity to AAV, anti-VEGF resistance, under treatment, variability of successful delivery or the location of the delivery. Potential reasons for variability between this Phase 2a study and the previous Phase 1 (Rakoczy et al., 2015) in BCVA and CPT outcomes were examined (Table S1). Product quality was evaluated and found to meet drug specifications in the lots used in both trials and showed no difference in biologic activity in vitro. Other possible causes include differing disease stage and anatomic appearance such as degree of subretinal fluid, geographic atrophy, subretinal fibrosis, pigment epithelial detachment, and CNV location. These variations in anatomic appearance and disease stage may also have impacted certain technical factors of therapy including Plerixafor Supplier in the retina and subretinal space. In the Phase 1 study, the baseline median BCVA in ETDRS letters was 40.0 (IQR: 33.0 to 54.0) versus 63.0 (IQR: 50.0 to 71.0) in the Phase 2a patients, while the median CPT was 549.0 (IQR: 268.0 657.0) compared to 332.5 (IQR: 295.5 to 459.8) μm, respectively. The difference in thickness could have had an impact on the mobility of sFLT-1 to the area of the subfoveal neovascularization via changing pharmacokinetic properties (Holash et al., 2002). Other possibilities are difficult to quantify but may include intrinsic VEGF load or the degree of anti-VEGF responsiveness in this cohort of patients. Additional preclinical laboratory studies evaluating potential sources of variation in the apparent clinical effect between the patients in this study and our initial report (Rakoczy et al., 2015) using this approach are underway. These include a determination of the optimal vector copy number to be injected, the impact of site of injection and the efficacy of alternative modified AAV vectors and cDNA constructs. There were several limitations to this study. These include the fact that this was an open label study as the gene therapy required a surgery (subretinal injection and vitrectomy) that could not be mimicked by a sham treatment due to the constraints of a clinical trial. Although the technicians measuring BCVA were masked, the open label study design allowed for potential patient or investigator bias. Also, the optimal method for subretinal administration has not been standardized relative to both safety and potential efficacy in this patient population with an anatomically abnormal macula due to intrinsic disease relative to the normal eye. The retreatment criteria differed slightly from other published studies (CATT Research Group et al., 2011; Lalwani et al., 2009), but were designed to allow for assessment of gene therapy activity as evidenced by reduced need for ranibizumab retreatments. There exist a number of different and evolving community standards for anti-VEGF retreatment including programmed monthly injections, treat and extend regimens, and PRN regimens. Furthermore, this Phase 2a study only assessed 1 dose of gene therapy against control, which prevented an understanding of a possible dose-response effect. Finally, 29 of 32 patients had previously been treated to varying degrees for wAMD, in contrast to most wAMD studies that have enrolled only treatment-naïve patients and that decision may have also affected the results of the trial.