Dose-Dependent Progression of Chorioretinal Atrophy at the Injection Site After Subretinal Injection of rAAV2/8 in Nonhuman Primates.

Objective: Progressive retinal atrophy has been described after subretinal gene therapy utilizing the adeno-associated virus (AAV) vector platform. To elucidate whether this atrophy is a consequence of inherent properties of AAV, or if it is related to the surgical trauma of subretinal delivery, we analyzed data from an Investigational New Drug-enabling study for PDE6A gene therapy in nonhuman primates. Design: Animal study (nonhuman primates), retrospective data analysis. Subjects: Forty eyes of 30 healthy nonhuman primates (macaca fascicularis) were included in the analysis. Two AAV dose levels (low: 1x10E11, high: 1x10E12) were compared with sham injection (balanced saline solution; BSS). Twenty untreated eyes were not analyzed. Methods: Animals were treated with a sutureless 23G vitrectomy and single subretinal injections of AAV.PDE6A and/or BSS. The follow-up period was 12 weeks. Atrophy development was followed using fundus autofluorescence (AF), OCT, fluorescence angiography, and indocyanine green angiography. Main Outcome Measures: Area [mm2] of retinal pigment epithelium atrophy on AF. Presence of outer retinal atrophy on optical coherence tomography. Area [mm2] of hyperfluorescence in fluorescence angiography and hypofluorescence in indocyanine green angiography. Results: Progressive atrophy at the injection site developed in 54% of high-dose-treated, 27% of low-dose-treated, and 0% of sham-treated eyes. At the end of observation, the mean ± SD area of atrophy in AF was 1.19 ± 1.75 mm2, 0.25 ± 0.50 mm2, and 0.0 ± 0.0 mm2, respectively (sham × high dose: P = 0.01). Atrophic lesions in AF (P = 0.01) and fluorescence angiography (P = 0.02) were significantly larger in high-dose-treated eyes, compared with sham-treated eyes. Rate of progression in high-dose-treated eyes was 4.1× higher compared with low-dose-treated eyes. Conclusion: Subretinal injection of AAV.PDE6A induced dose-dependent, progressive retinal atrophy at the site of injection. Findings from multimodal imaging were in line with focal, transient inflammation within the retina and choroid and secondary atrophy. Atrophic changes after gene therapy with AAV-based vector systems are not primarily due to surgical trauma and increase with the dose given. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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

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.

An Optimized Treatment Protocol for Subretinal Injections Limits Intravitreal Vector Distribution.

Purpose: Subretinal injections (SRis) are commonly used in retinal gene therapy procedures to deliver adeno-associated virus (AAV) to photoreceptors and retinal pigment epithelial cells. We present an optimized surgical protocol to minimize off-target application of AAV in the vitreous, which in turn reduces the risk of extensive biodistribution and inflammation, ultimately leading to enhanced safety of the therapy. Design: Experimental animal research study. Participants: Eight cynomolgus monkeys (Macaca fascicularis). Methods: Subretinal injections with an AAV2/8 vector were performed. The animals were allocated to 2 different vector dose groups (1×10ˆ 11 and 5×10ˆ 11 viral genomes [vg]). Samples of intravitreal fluid were taken at the end of the SRi procedure and again after a 3-minute lavage (wash-out) with balanced salt solution (BSS). Main Outcome Measures: Intravitreal vector genome copies were analyzed with quantitative polymerase chain reaction and compared between groups. Results: Even uneventful SRi leads to dissemination of millions of AAV particles (0.1-0.7% of viral vector loading dose) into the vitreous cavity. Three minutes of lavage led to a substantial decrease (on average 96%) of intravitreal vector load. Conclusions: Multiple studies have shown that the intravitreal space is not as immune privileged as the subretinal space. Intravitreal AAV particles disseminate into the bloodstream, lead to increased biodistribution into lymphatic tissue, and help to stage an immune response with implications for both safety and efficacy. Therefore, minimizing off-target vector application after reflux of vector from the subretinal space is of significant interest. We show that a simple lavage of intravitreal fluid efficiently decreases the intravitreal vector load. Such a step should be considered when performing subretinal gene therapy.

Subretinal and Intravitreal Retinal Injections in Monkeys.

Ocular anatomy and physiology in cynomolgus monkeys (Macacca fascicularis) are very similar to that found in the human visual system. Therefore and despite significant ethical and economical implications, these animals constitute an excellent model system for toxicology and biodistribution studies in the development of new and meaningful treatment strategies for ocular disorders. The methods for delivery of investigational new drugs (INDs) to the ocular tissue are virtually identical to the methods used in clinical practice. This protocol explains in detail the method of applying INDs to the vitreous cavity or the subretinal space in monkeys.

Subretinal Injection for Gene Therapy Does Not Cause Clinically Significant Outer Nuclear Layer Thinning in Normal Primate Foveae.

Purpose: Despite ever-growing adoption of subretinal (SRi) and intravitreal injections (IVTi) in ocular gene therapy trials, concerns regarding possible deleterious effects of the SRi on the outer retina are yet to be addressed. SRi offers several advantages over IVTi, such as a better photoreceptor transduction efficiency and a limited off-target exposure. We assessed structural changes in the outer retina in nonhuman primates following either SRi or IVTi of a gene therapeutic or control solution and compared both techniques in a noninferiority analysis. Methods: In a toxicology study, 22 cynomolgus monkeys underwent single intraocular injections with rAAV2/8 or vehicle; 18 animals received SRi, 4 animals received IVTi. Outer nuclear layer (ONL) thickness change on optical coherence tomography was used for a noninferiority analysis. Preservation of the physiological foveal bulge was used as a secondary outcome measure. Results: The average ONL change from baseline after 2 weeks was -6.54 ± 5.16 (mean ± SD µm) and +1.50 ± 4.36 for SRi and IVTi groups accordingly. At 13 weeks, the SRi group maintained a difference of -6.54 ± 9.66 while IVTi group gained +1.00 ± 4.24. The ellipsoid zone line was transiently lost after SRi and completely recovered by 13 weeks in 77% of eyes. One SRi case resulted in subfoveal pigment accumulation and 39% ONL thinning. Conclusions: Despite limited ONL thinning following SRi, the observed effect was under the predefined clinical significance threshold. The SRi has proven not to be inferior to the IVTi in terms of ONL thickness loss and estimated loss of visual acuity.

Retinal Gene Therapy: Surgical Vector Delivery in the Translation to Clinical Trials.

An exceptionally high number of monogenic disorders lead to incurable blindness, making them targets for the development of gene-therapy. In order to successfully apply therapeutic vector systems in vivo, the heterogeneity of the disease phenotype needs to be considered. This necessitates tailored approaches such as subretinal or intravitreal injections with the aim to maximize transduction of target cell populations, while minimizing off-target effects and surgical complications. Strategic decisions on parameters of the application are crucial to obtain the best treatment outcomes and patient safety. While most of the current retinal gene therapy trials utilize a subretinal approach, a deeper understanding of the numerous factors and considerations in choosing one delivery approach over the other for various ocular pathologies could lead to an improved safety and treatment efficacy. In this review we survey different vector injection techniques and parameters applied in recent retinal (pre-)clinical trials. We explore the advantages and shortcomings of each delivery strategy in the setting of different underlying ocular pathologies and other relevant factors. We highlight the potential benefits for patient safety and efficacy in applying those considerations in the decision making process.