Berger, Aubrey

Subretinal and intravitreal injection of retinal gene therapy is associated with serious adverse events and poor efficacy. We sought to improve retinal gene therapy delivery by developing fibrin hydrogel encapsulated adeno-associated virus (FE-AAV). Here we investigate conditions for storage and stability for FE-AAV. FE-AAV containing 1.9 × 109 genome copies of AAV2/2-CMV-GFP was manufactured using fibrinogen reconstituted in 0.01 M sodium citrate, pH 7 (NaC) or phosphate-buffered saline containing 0.001% (v/v) Pluronic F68 (F68). Samples were stored at either −80 °C or 4 °C for up to 16 weeks. Changes in transduction efficiency, and mechanical and physical properties were evaluated. In vitro transduction was significantly (p < 0.05) reduced for FE-AAV manufactured with NaC. In contrast, we observed no change in transduction through 16 weeks for FE-AAV made with F68. Physical changes occurred in FE-AAV stored at −80 °C. In contrast to FE-AAV formulated with NaC, FE-AAV formulated with F68 and stored at 4 °C for 16 weeks was essentially equivalent to freshly made FE-AAV and retained the ability to transduce retinal pigment epithelial (RPE) cells in the pig eye. We conclude that FE-AAV formulated with F68 and stored at 4 °C is stable and shows potential for retinal gene therapy for at least 4 months following manufacture.

Death or dysfunction of retinal pigment epithelium (RPE) cells occurs in age-related macular degeneration (AMD) and certain inherited retinal dystrophies (IRDs). Induced-pluripotent stem cell (iPSC) derived-RPE have been used in early-stage clinical trials to treat AMD and IRDs by injecting them as a cell suspension or monolayers. While RPE transplant shows therapeutic potential, issues ranging from failure to repopulate the entire treatment area, clumping and monolayer folding, and a foreign body response to the support have been reported. We’ve shown that RPE can be grown on high concentration (&gt;30 mg/mL) degradable fibrin hydrogels, and that cell free fibrin hydrogels implanted in the subretinal space degrade without causing inflammation. Here we describe manufacture and surgical implantation of degradable fibrin hydrogels carrying iPSC-RPE into a porcine model of geographic atrophy (GA). Large (15.25 × 58.42 × 0.2 mm) fibrin gel blanks were produced by injection molding, and iPSC-RPE were grown on their surface. Using a mechanical punch, the blank was subdivided into 1.5 × 5.0 × 0.2 mm doses, which fit a custom tool used for storage and surgical placement. Following aseptic packaging, RPE and gels were stable at 37 °C for at least 7 weeks. When transplanted into a pig model of GA, the fibrin scaffold degraded in &lt;1 month and the iPSC-RPE provided partial rescue from GA as assessed by preservation of photoreceptors and blood flow in the choriocapillaris. We conclude that iPSC-RPE delivered on degradable fibrin hydrogels represent a potentially safe and effective approach to RPE transplantation.

Subretinal injection of adeno-associated virus (AAV) is generally more efficacious and less inflammatory than intravitreal injection for retinal gene therapy. However, adverse events (e.g., chorioretinal atrophy) have been reported in many patients receiving subretinal injection of Luxturna (voretigene neparvovec-rzyl) and experimental gene therapies. Subretinal injection confines transduction to the treated area. To address this, we manufactured high-concentration fibrin hydrogels encapsulating AAV2–green fluorescent protein (AAV2- GFP ). Gels had homogeneous AAV distribution, desired mechanical properties, and retained infectivity. Epiretinal placement of fibrin–AAV2- GFP ( n = 11) was compared to subretinal ( n = 5) and intravitreal AAV2- GFP injection ( n = 3). The subretinal group exhibited inconsistent retinal pigment epithelium (RPE) transduction restricted to the injection region with severe atrophy in two cases. The intravitreal group had weak transduction and inflammation. In contrast, epiretinal hydrogels degraded within days and led to broad transduction of RPE without atrophy or inflammation. This technology could advance gene therapy for retinal degenerations and other ocular or systemic disorders.

ABSTRACT Background To explore the prevalence of serious adverse events ( SAEs ) associated with retinal viral gene therapy and to examine trends influencing SAE occurrences in human gene therapy surgeries and pre‐clinical animal trials. Methods Literature review was performed to identify peer‐reviewed human and animal studies relevant to viral gene therapy, subretinal injections, and intravitreal injections. For clinical trials and post‐approval LUXTURNA studies, only those that examined SAEs were included. Results Of included clinical trial studies ( n = 31), SAEs were recorded in 51 out of 438 eyes (11.6%) that received subretinal injections and in 11 out of 348 eyes (3.2%) that received intravitreal injections. There were fewer intravitreal‐related SAEs and less vision loss compared to subretinal gene therapy. Inflammation was the predominant SAE following intravitreal injections, whereas unexplained vision loss was the most common for subretinal injections. Clinical trials utilising subretinal injections met primary or secondary efficacy endpoints more than intravitreal trials. Eighteen studies (429 eyes) of post‐approval LUXTURNA were reviewed, and SAEs were reported in 24.7% of eyes, retinal degeneration being most common (20.7%). For 58 animal studies, SAEs were recorded in 17.3% of eyes that received subretinal injections and 8.7% of eyes that received intravitreal injections. Conclusions Subretinal injections show higher efficacy than intravitreal injections but are associated with more serious adverse events. Consistent adverse event patterns in humans and large animals highlight their predictive value for safety. There is a need for optimised delivery methods, refined dosing protocols, and improved post‐treatment monitoring to improve safety and effectiveness in gene therapy.