Long-Term Safety Evaluation of Continuous Intraocular Delivery of Aflibercept by the Intravitreal Gene Therapy Candidate ADVM-022 in Nonhuman Primates

Abstract

Purpose: To evaluate the long-term safety of vascular endothelial growth factor (VEGF) suppression with sustained aflibercept expression after a single intravitreal injection (IVI) of ADVM-022, an anti-VEGF gene therapy, in non-human primates (NHPs).

Methods: Non-human primates received bilateral IVI of ADVM-022, a gene therapy vector encoding aflibercept, a standard of care for the treatment of VEGF-based retinal disease. Aflibercept levels from ocular fluids and tissues were measured. Ocular inflammation was assessed by slit lamp biomicroscopy and fundoscopy. The integrity of the retinal structure was analyzed by optical coherence tomography and blue light fundus autofluorescence and electroretinography was performed to determine retinal function. Histologic evaluation of the retina was performed at the longest time point measured (2.5 years after injection).

Results: Sustained expression of aflibercept was noted out to the last time point evaluated. Mild to moderate inflammatory responses were observed, which trended toward spontaneous resolution without anti-inflammatory treatment. No abnormalities in retinal structure or function were observed, as measured by optical coherence tomography and electroretinography, respectively. RPE integrity was maintained throughout the study; no histologic abnormalities were observed 2.5 years after ADVM-022 IVI.

Conclusions: In non-human primates, long-term, sustained aflibercept expression and the resulting continuous VEGF suppression by a single IVI of ADVM-022, appears to be safe, with no measurable adverse effects on normal retinal structure and function evaluated out to 2.5 years.

Translational relevance: Together with the results from previous ADVM-022 preclinical studies, these data support the evaluation of this gene therapy candidate in clinical trials as a potential durable treatment for various VEGF-mediated ophthalmic disorders.

Keywords: ERG; OCT; adeno-associated viral vector; aflibercept; anti-VEGF.

Figure 1.

A single IVI of ADVM-022 results in sustained levels of aflibercept in the vitreous humor throughout period out to 30 months that are comparable to the levels of aflibercept in the terminally collected ocular tissues. (A) Levels of aflibercept were quantified from the vitreous humor samples collected at indicated time points. (B) Levels of aflibercept were quantified from the terminally collected ocular tissues (retina, choroid, ciliary body, aqueous, and vitreous humor). Aflibercept levels were measured by VEGF-165 anti-human Fc sandwich ELISA assay. Green bars show mean levels of aflibercept. A055 OD sample showed no measurable levels of aflibercept in choroid. Levels of aflibercept in vitreous and aqueous humor in Figure 1B are expressed as µg/g based on the assumption that density of the tissues is approximately 1 g/mL. Please note that the error bars are smaller than the symbol. The values reported are above the lower limit of quantification.

Figure 2.

(A–D) Ocular clinical pathology and (E) IOP in the individual eyes treated with ADVM-022 were assessed throughout the course of two studies at the indicated time points and out to 30 months after IVI. Examinations revealed mild inflammation with a resolving trend throughout the time course. The parameters scored by the Hackett–McDonald irritation and inflammation scoring system are shown: (A) keratic precipitates, (B) vitreous cells, (C) aqueous cells, (D) lens capsule deposits, and (E) IOP. No vitreous haze has been detected except in one eye (A055, OD) at only one time point (12.5 months) at grade 1+. (E) The IOP was within the normal range except for both eyes in animal K700 on D14 (when it decreased to 3.3 mm Hg). The IOP in both eyes of K700 returned to normal at the next time point assessed (day 28). Normal IOP lower and upper IOP limits were determined based on the IOP measurements conducted on 148 eyes at baseline. The lower and upper IOP limits are designated as 2 standard deviations of the mean value (µ ± 2σ). Scores are represented for each eye in the study for every time point assessed and each symbol represents one individual eye.

Figure 3.

Long-term expression of aflibercept in the retina from ADVM-022–treated animals does not affect retinal morphology features as evaluated by the OCT or H&E staining. (A) OCT images of both eyes from a representative animal (K700) treated with ADVM-022 were analyzed at baseline and at 30 months after treatment. (B) RPE and (C) ONL-PR1-PR2 layer thickness measurement revealed no difference in any of the segments analyzed. Analysis is performed at baseline and at respective terminal time points (21 or 30 months after dosing) (Student t test, P = 0.99 for RPE analysis and P = 0.79 for ONL-PR1-PR2 analysis). Please note that some of the error bars are smaller than the symbol size. (D) FAF micrographs of animal K700 at baseline and 30 months after ADVM-022 treatment. (E) H&E staining of animal K700 OS retinal tissue 30 months after treatment. Note the integrity of all retinal layers and the absence of any abnormality in the morphology.

Figure 4.

ERG analysis of the ADVM-022 treated animals at the late time points reveals normal ERG responses. (A) Scotopic ERG recordings were performed on 2 animals (A055 and A075, n = 4 eyes) treated with ADVM-022 19 months after injection and compared with vehicle-treated animals (n = 6 eyes). (B) Analysis of scotopic A-wave and (C) B-wave revealed no statistically significant difference between ADVM-022 and vehicle treated animals (repeated measures ANOVA A-wave P = 0.95, B-wave P = 0.48). (D) First order three-dimensional multifocal ERG topography density at 30 months after treatment (K700) revealed normal recordings. The stimulus configuration is an array of 103 hexagons scaled with retinal eccentricity. A local response was derived for each of the 103 stimulus elements. First-order ERG recording traces are shown in the lower panels. The multifocal ERG traces are shown equally spaced for clarity. (E) ERG response density at different regions of retina from both eyes for the animal treated with ADVM-022 (K700) at 30 months after the dose were plotted against the distribution of the ERG responses in treatment-naïve, healthy animals (white boxes, n = 20 eyes) and were within the limits of responses recorded in the treatment-naïve animals.