Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals.

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Libechov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Mitochondrial Dysfunction in a High Intraocular Pressure-Induced Retinal Ischemia Minipig Model.

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI.

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs.

PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.