Suprachoroidally Delivered DNA Nanoparticles Transfect Retina and Retinal Pigment Epithelium/Choroid in Rabbits

Abstract

Purpose: This study evaluated ocular tolerability and transfectability of nonviral DNA nanoparticles (DNPs) after microneedle-based suprachoroidal (SC) administration, in comparison to subretinal (SR) administration.

Methods: The DNPs consisted of a single copy of plasmid DNA with a polyubiquitin C/luciferase transcriptional cassette compacted with 10 kDa PEG-substituted lysine 30-mer peptides (CK30PEG10k). New Zealand White rabbits (n = 4 per group) received a unilateral SC injection (0.1 mL via a microneedle technique) of ellipsoid-shaped DNPs, rod-shaped DNPs, or saline (negative control). A cohort of rabbits (n = 4) also received a single unilateral SR injection (0.05 mL via a transvitreal approach) of rod-shaped DNPs. At day 7, luciferase activity was measured in the retina and retinal pigment epithelium (RPE)-choroid via bioluminescence assay. A cohort of rabbits received a SC injection of analogous DNPs to assess spread of DNP injectate in the suprachoroidal space (SCS) via optical coherent tomography and histology.

Results: Suprachoroidal injection of DNPs resulted in reversible opening of the SCS circumferentially and posteriorly and was generally well tolerated, with no significant ocular examination score changes, intraocular pressure abnormalities, or changes in electroretinography amplitudes on day 7 compared to the baseline. High luciferase activity was observed in the retina and RPE-choroid of eyes that received SC DNPs (rod and ellipsoid shape) and SR DNPs (rod shape) compared to controls. The mean luciferase activity in RPE-choroid and retina was comparable between SC and SR administrations. Transfection in the RPE-choroid was approximately 10-fold higher than in the retina after either SC or SR administration of DNPs.

Conclusions: Suprachoroidal and SR administration of DNPs resulted in comparable transfection of retina and RPE-choroid.

Translational relevance: Suprachoroidal delivery of DNPs offers the potential to precisely target chorioretinal tissues while avoiding surgical risks associated with SR injection, and it may offer an office-based nonsurgical gene therapy option for the treatment of retinal diseases.

Keywords: DNA nanoparticles; chorioretinal diseases; nonviral gene therapy; suprachoroidal delivery.

Figure 1.

Transmission electron micrographs of rod-shaped (A) and ellipsoid-shaped (B) pEUL DNA NPs manufactured for the rabbit studies. The black bar scale in each photo represents 200 nm.

Figure 2.

Total ocular exam scores over time. Ocular exams involved conjunctival redness/congestion, chemosis, discharge, corneal opacity, cornea pannus, aqueous flare, pupillary light reflex, cellular flare, lens, ocular fundus, vitreous body, optic nerve head, retinal vasculature and choroidal pathology via slit-lamp, direct and indirect ophthalmoscope, and ocular fundus photography. Ocular examination (OE) scores were comparably low (0–1) for suprachoroidally administered DNPs (rod and ellipsoid shape) and control (saline-injected) eyes. OE scores were significantly higher (P = 0.001) for subretinally administered DNPs (rod shape) on day 1 and similar on day 7 compared to untreated eyes.

Figure 3.

Intraocular pressure (mmHg) from dosed (left) eyes (A) and undosed (right) eyes (B) over time. IOP was measured in triplicate per eye per time point. Mean IOP values were within the normal variability of measurements for all groups except on day 1, when rod-shaped DNPs were administered subretinally (P = 0.0159). No statistical difference was observed in mean IOP values among all different groups in the untreated eyes or saline-treated eyes.

Figure 4.

Average scotopic b-wave amplitude (A) and photopic b-wave amplitude (B) over time. No statistically significant difference was observed among various groups analyzed (p = ns; a one-way ANOVA model with treatment group as the fixed effect with Bonferroni's multiple comparison tests to assess statistical significance between groups).

Figure 5.

Representative optical coherent tomography images of rabbit eyes at baseline (A), immediately after SC injection on the day of dosing (B), and on day 30 postinjection (C). Double arrows indicate expansion of suprachoroidal space. ch, choroid; ONH, optic nerve head; ret, retina; s, sclera; SCS, suprachoroidal space. Images from the same quadrant of the same eye have been selected for longitudinal assessment.

Figure 6.

Representative histology images (hematoxylin and eosin stains) of rabbit eyes on day 7 postdose. (A) SC ellipsoid DNPs (100×): No intraocular inflammation. Mild to moderate infiltration of mononuclear cells noted in choroid near sclera. (B) SC ellipsoid DNPs (400×): Mild, multifocal areas of choroidal thickening, mononuclear inflammation; no intraocular inflammation visible. (C) SC rod DNPs (200×): No intraocular inflammation. (D) SC rod DNPs (400×): Expanded suprachoroidal space containing tan material (likely artifact). No inflammation or degeneration of adjacent tissue visible. (E) SR rod DNPs (400×): Focal area of retinitis and retinal separation, with a moderate area of retinal degeneration superior to the optic nerve. (F) SR rod DNPs (600×): Moderate to severe retinal and subretinal inflammation at site of SR injection superior to optic nerve. Asterisks represent infiltrated mononuclear cells. C, choroid; R, retina; S, sclera; V, vitreous humor.

Figure 7.

Luciferase activity in the RPE-choroid (A) and retina (B) of rabbit eyes dosed DNPs either subretinally (SR) with rod-shaped DNPs or suprachoroidally with both rod- and ellipsoid-shaped DNPs. Undosed eyes and saline-injected eyes were negative controls. One-way ANOVA with Bonferroni's multiple comparison test (GraphPad Prism). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant.