Longitudinal Evaluation of Hyper-Reflective Foci in the Retina Following Subretinal Delivery of Adeno-Associated Virus in Non-Human Primates

Abstract

Purpose: The purpose of this study was to evaluate whether clinical grade recombinant adeno-associated virus serotype 8 (rAAV8) leads to increased appearance of hyper-reflective foci (HRF) in the retina of non-human primates (NHPs) following subretinal gene therapy injection.

Methods: Different doses of rAAV8 vector (rAAV8. human phosphodiesterase 6A subunit (hPDE6A) at low dose: 1 × 1011 vector genomes (vg), medium dose: 5 × 1011 vg, or high dose: 1 × 1012 vg) were injected subretinally into the left eyes of NHPs in a formal toxicology study in preparation of a clinical trial. Right eyes received sham-injection. After 3 months of in vivo, follow-up retinal sections were obtained and analyzed. The number of HRF on spectral domain-optical coherence tomography (SD-OCT) volume scans were counted from both eyes at 30 and 90 days.

Results: Animals from the high-dose group showed more HRF than in the low (P = 0.03) and medium (P = 0.01) dose groups at 90 days. There was a significant increase in the mean number of HRF in rAAV8-treated eyes compared with sham-treated eyes at 90 days (P = 0.02). Sham-treated eyes demonstrated a nonsignificant reduction of HRF numbers over time. In contrast, a significant increase over time was observed in the rAAV8-treated eyes of the high dose group (P = 0.001). The presence of infiltrating B- and T-cells and microglia activation were detected in rAAV8-treated eyes.

Conclusions: Some HRF in the retina appear to be related to the surgical trauma of subretinal injection. Although HRF in sham-treated retina tends to become less frequent over time, they accumulate in the high-dose rAAV8-treated eyes. This may suggest a sustained immunogenicity when subretinal injections of higher doses of rAAV8 vectors are applied, but it has lower impact when using more clinically relevant doses (low and medium groups).

Translational relevance: An increase or persistence of HRFs following retinal gene therapy may indicate the need for immunomodulatory treatment.

Figure 1.

SD-OCT image of HRF in a sham-treated eye of NHP. The 30 degrees fundus IR image with overlying position of B-Scan (left panel, green line) and cross-sectional SD-OCT B-scan (right panel) demonstrate the appearance of HRF in the ONL (arrow heads) within the bleb area (white dashed line) 30 days after subretinal injection. HRF, hyper-reflective foci; IR, infrared; NHP, non-human primate; ONL, outer nuclear layer; SD-OCT, spectral-domain optical coherence tomography.

Figure 2.

Representative distribution maps of HRF in the retina of NHPs 30 and 90 days after subretinal injection of BSS (sham) or different doses of rAAV8. HRF are plotted on IR and FAF images in different colors. Green dots = HRF were identified by three observers; yellow dots = HRF were identified by two observers; red dots = HRF were identified by one observer. FAF, fundus autofluorescence; Group 1, low-dose group; Group 2, medium-dose group; Group 3, high-dose group; HRF, hyper-reflective foci; IR, infrared; NHP, non-human primate.

Figure 3.

Kinetics of HRF of over time in sham- (a) and rAAV8-treated (b) eyes. Data are shown as mean ± standard deviation for the 3 groups at days 30 and 90 post-subretinal injection. Each colored symbol represents one animal (4 per group, P value = ≤0.01: **). Graphic representation of the percentage of mean change in the number of HRF normalized to 30-days measurements (c). Values below 0 indicate a decrease in the percentage of the number of HRF from day 30 to day 90; and values above 0 indicate an increase of HRF. Group 1, low-dose group; Group 2,medium-dose group; Group 3, high-dose group; HRF, hyper-reflective foci; ONL, outer nuclear layer.

Figure 4.

Thickness of ONL over time in sham- (a) and rAAV8-treated (b) eyes. Data are shown as mean ± standard deviation for the 3 groups at days 30 and 90 post-subretinal injection. Each colored symbol represents one animal (4 per group, P values = ≤0.05: *; ≤0.001: ***). Group 1, low-dose group; Group 2, medium-dose group; Group 3, high-dose group; ONL, outer nuclear layer.

Figure 5.

Immunohistochemical analysis of sham- (a–l) and rAAV8-treated (m–x) eyes from group 3. The 60 degrees fundus IR image showing the representative HRF distribution maps (a, m) with overlying position of B-scan (green line) and cross-sectional SD-OCT B-scans (b, n) demonstrate the appearance of HRF within the treated area 90 days after subretinal injection. Normal H&E staining of an area close to the SD-OCT scan (c, o). Immunohistochemical permanent staining of RPE65 shows the integrity of the RPE layer (d, p). B- and T-cells (CD20⁺ and CD3⁺ cells, respectively) were not detected in the sham treated eyes (e–h) but they were found in the rAAV8 treated eyes (q–t). The macrophage marker CD68 was not detected neither in the sham- (j) nor in rAAV8-treated (v) eyes. However, microglia cells were more active in rAAV8-treated eyes (w) than in sham-treated eyes (k). Scale bar: 50 µm. HRF, hyper-reflective foci; H&E, hematoxylin and eosin; IR, infrared; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium; SD-OCT, spectral-domain optical coherence tomography.

Figure 6.

Immune retinal infiltrates found in perivascular (a–j) and subretinal (k–t) areas of animals from group 3 and group 1, respectively. H&E staining shows the appearance of leukocytic cells infiltration in the perivascular area of a retinal blood vessel a (black arrow) and between the neural retina and the choroid k (black arrow). RPE65 staining shows the state of the RPE layer f and p being completely absent in the area where the subretinal infiltrate is present p. CD20⁺ and CD3⁺ cells b to e and l to o together with microglia activation i and s were found in both types of infiltrates. The macrophage marker CD68 was only detected in the perivascular infiltrate h. Scale bar: 30 µm. H&E, hematoxylin and eosin; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium.