185. Safety Study by Validated Immunoassays in a Phase III Study of Subjects with Inherited Retinal Dystrophy Due to Mutations in the Gene Encoding Human Retinal Pigment Epithelium-Specific Protein 65 (RPE65) Injected with Adeno-Associated Viral Vectors
Authors: Daniel J. Hui, Yifeng Chen, Thomas Antrilli, Yun Liu, Romuald Corbau, Stephen R. Russell, Albert M. Maguire, Jean Bennett, Katherine A. High
Journal: Molecular Therapy (2016)

Mutations in the gene encoding Retinal Pigment Epithelium-Specific Protein 65 (RPE65) cause impaired vision from an early age and eventually total vision loss later in life. Recent work by our group and others demonstrated the use of SPK-RPE65, an adeno-associated viral (AAV) vector, to deliver the gene stably in small and large animal models, while Phase 1 and Phase 3 clinical trials with SPK-RPE65 showed promising results (Maguire et al., 2008; Maguire et al., 2009; Simonelli et al., 2010; Maguire, AAO 2015). The Phase 3 trial was an open-label, randomized study using a subretinally-delivered AAV2 vector to augment the RPE65 gene. Thirty one subjects were enrolled from centers at The Children's Hospital of Philadelphia or University of Iowa. Twenty of the 21 intervention group subjects received 1.5E11 vg non-simultaneous injections to each eye. In addition to a separate efficacy assessment that measured the primary and secondary endpoints for the trial, an independent safety study was performed that included previously established immunological assays designed to monitor cellular immune responses. Two assays were subjected to further validation in-house and used for immune analysis of clinical samples obtained from all intervention subjects. An Enzyme-Linked Immunosorbent Assay (ELISA), capable of detecting a titer of at least 1.55 ug/mL anti-AAV2-capsid human IgG, was performed on 21 subjects covering up to 4 timepoints (baseline prior to injection, day 30, day 90 and 1 year). To better evaluate the results, the subjects were placed into 6 categories based on their antibody titer profile (Table 1).Table 1Anti-AAV2 Profile of Intervention Group (n=21)Number of SubjectsAnti AAV Titer Range (ug/mL)Anti-AAV2 Profile7<1.55Below quantification limit31.72 – 2.91Low pre-existing antibody titer216.37 – 19.61Moderate pre-existing antibody titer454.39 – 248.55High pre-existing antibody titer41.73 – 87.02Antibody titer developed after vector administration1N/AWithdrew Open table in a new tab The other immunoassay performed on the intervention group was an interferon-γ Enzyme-Linked Immunospot Assay (ELISPOT) to assess T cell responses against AAV2 capsid or RPE65 transgene product. The same set of intervention subjects/timepoints was analyzed with positive T cell responses defined as ≥50 spot forming units (SFU) and 3-fold the background (media) control for AAV2 and greater than the statistically determined cutoff of 161.3 SFU for RPE65. Eighteen of the 21 intervention subjects tested negative for T cell responses against AAV2 and RPE65 across all timepoints. One subject was positive against AAV2 capsid at baseline (55.0 SFU) and positive against RPE65 at the 1 year timepoint (171.7 SFU). Another subject was positive at 1 year for RPE65 only (170.0 SFU). These positive responses were considered very weak with respect to threshold cutoff values. One subject displayed a moderate response (518.3 SFU) against RPE65 at baseline only, while all subsequent timepoints were negative. The positive T cell responses observed at baseline prior to vector administration are unlikely to be related to gene transfer. Considering the cumulative immune response data collected from the two validated immunoassays performed here, the Phase 3 trial provided results supporting the immunologic tolerability of the delivery of AAV2 vector to the subretinal space in the eye to treat inherited retinal dystrophy due to mutations in RPE65. Efforts are ongoing to correlate these findings with available efficacy data.