Analysis of anti-AAV2 and anti-RPE65 Nab and responses over time after initial injection (bold) and after readministration. The exact time points evaluated differed for the initial and the FO study (table S1). There were no detectable anti-RPE65 Nabs detected after the initial injection (18). However, these data are not included in Table 2 because the assay was modified for the FO study measurements. Results are indicated as reciprocal dilutions of serum samples (see Supplementary Methods). Anti-AAV2 titers after the first injection were previously reported (18) and are shown here for comparison with the FO titers. The titers remained low throughout the course of the study, with a minor increase at week 8 for CH12 (italicized) followed by a return to baseline. High FO baseline NAbs directed against RPE65 protein were detectable in subjects CH12 and CH11. The positivity may have been due to cross-reaction with another RPE65-like protein or that the subject may produce a dysfunctional but immunologically detectable protein. The positive responses detected early on decreased slightly over time. NA, sample not available.