Results br Discussion This Phase

Results
Discussion This Phase 2a study of 32 patients is the largest ocular AAV clinical trial to date and serves to confirm the results of the Phase 1 study (Rakoczy et al., 2015), which showed that subretinal injection of rAAV.sFLT-1 is safe. The study also investigates the potential association between the safety and patient-focused efficacy of rAAV.sFLT-1 gene therapy delivered subretinally. Results of the study showed no serious gene therapy-related ocular or systemic side effects. All AEs related to gene therapy or study procedure were mild or moderate and resolved without sequelae. These findings were consistent with the Phase 1 study (Rakoczy et al., 2015), as well as other AAV ocular studies (Vasireddy et al., 2013; Maguire et al., 2008; Pierce and Bennett, 2015; Simonelli et al., 2010; Testa et al., 2013). The majority of mild and moderate ocular AEs were associated with the surgery involving vitrectomy and subretinal injection (Maguire et al., 2008; Rakoczy et al., 2015). As these procedures were a necessary component of the AAV therapy, they are an important consideration for safety. A variety of ocular hemorrhages associated with the surgery were mild in nature without any visual significance and with no permanent sequelae. Cataracts are a known side effect of vitrectomy (Blankenship and Machemer, 1985) and progression to visually significant nuclear cataract in all phakic gene therapy patients and in 1 control patient was noted. It is not yet clear if subretinal injection can be done safely without vitrectomy. A higher number of systemic AEs were reported in the gene therapy group compared to the control group, which we interpret as due to the reporting procedures described in the Methods section. Respiratory system-related AEs were reported more frequently in patients treated with rAAV.sFLT-1 (28%) compared to controls (18%). However, the infections were heterogeneous in character, and differences in frequency between the gene therapy group and control group were not statistically significant. Overall the incidence of respiratory system-related AEs in the gene therapy group was less frequent than rates published for existing anti-VEGF agents in late-stage wAMD trials, so an association with rAAV.sFLT-1 gene therapy seems unlikely. Results of rAAV.sFLT-1 qPCR, AAV2 Anisomycin ELISA, and sFLT-1 protein quantitation indicate that the biodistribution of rAAV.sFLT-1 outside the target tissue (retina) after subretinal injection is limited and transient. No rAAV.sFLT-1 DNA or AAV2 capsid were detected in any of the assessed samples at or after the week 4 visit. The measured systemic levels of sFLT-1 and VEGF in serum, urine, and saliva were highly variable, fluctuating and dependent on the individual and we could not identify any trend that would have suggested systemic effect. The only immune response to rAAV.sFLT-1 therapy detected was sero conversion of nAbs observed in 3 patients which is consistent with previously published reports showing that subretinal administration of AAV does not frequently induce humoral immune responses to the AAV capsid (Li et al., 2008). Recent reports show that subretinal delivery of rAAV does not affect the efficacy of rAAV.sFLT-1 therapy when subsequently administered to the fellow eye (Li et al., 2008; Bennett et al., 2012). Amongst the patients who received the rAAV.sFLT-1 gene therapy, 12 (57·1%) had pre-existing nAbs to AAV2 and it seems that pre-existing nAbs to AAV2 do not necessarily decrease the efficacy of the gene therapy administered by subretinal injection. This study does not raise any new concerns for ocular gene therapy with respect to the variables of patient safety and immune response and confirms the published work of others (Bennett et al., 2012). Unlike many of the previously reported anti-VEGF trials (CATT Research Group et al., 2011; IVAN Study Investigators et al., 2012; Heier et al., 2012) for wAMD, 29 of 32 (90·6%) recruited patients in this study were not treatment-naïve, having received a median of 9.0 (IQR: 5.0 to 14.0) previous anti-VEGF injections. The chronic nature of baseline disease in the patients participating in this safety trial may have diminished the potential for large vision gains or fluid loss on SD-OCT. In addition the study was designed to assess the safety of rAAV.sFLT-1 delivered by subretinal injection and sought to provide PRN ranibizumab therapy in both arms. Therefore, no significant difference in BCVA and CPT between the gene therapy and control groups was expected. Nevertheless, the median change in BCVA for the gene therapy group was +1·0 ETDRS letters at week 52, and for the control group was −5·0 ETDRS letters confirming that rAAVsFLT-1 did not have a deleterious effect. In the control group, this loss of ETDRS letters potentially could suggest under-treatment of the wAMD with ranibizumab, greater intrinsic disease activity or a higher complication rate for intravitreal injections. However, closer examination reveals that this loss was due to 3 of 11 (27·3%) patients who lost in excess of 20 ETDRS letters caused by infrequent but well known complications of the natural history of wAMD and PRN intravitreal therapy. Once these 3 patients were removed from the analysis there was no statistically significant difference in BCVA improvement between the gene therapy and the control groups. The incidence of moderate vision loss was similar in the treatment and control groups suggesting that there might be a subgroup of patients who do not have the capacity to respond to anti-VEGF treatments. As all the phakic patients in the gene therapy group required cataract surgery presumed secondary to the initial vitrectomy, lens status and cataract removal were investigated as possible explanations for the median final BCVA of this group being higher than the control group. Although the numbers are small, post hoc analysis of initial lens status and need for cataract surgery was not found to drive the BCVA in the rAAV.sFLT-1-treated group and the control group at the week 52 time point.