Suprachoroidal and Subretinal Injections of AAV Using Transscleral Microneedles for Retinal Gene Delivery in Nonhuman Primates

Abstract

Retinal gene therapy using adeno-associated viruses (AAVs) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produce a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space, a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but it elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the bloodretinal barrier.

Host immune responses after suprachoroidal delivery of AAV8 in nonhuman primate eyes

“…These results are consistent with our prior study which demonstrated higher concentrations of serum NAbs from intravitreal than suprachoroidal or subretinal AAV8 as measured using an in vitro transduction inhibition assay. 21 By contrast, only animals that received suprachoroidal AAV8 developed anti-GFP antibodies, which reached the highest levels at month 3, while the animal that received only intravitreal AAV8 did not ( Figure 3B). As Rhesus 02 received high-dose suprachoroidal AAV8 in both eyes, we further validated the humoral response to GFP by performing flow cytometry on peripheral blood mononuclear cells (PBMCs) collected from the serum of this animal, and found expansion of GFP-responsive plasma B-cells (CD19-,CD27+,CD38+, HLADRlow) after suprachoroidal AAV8 injection ( Figure 3C, Supplementary Figure 1) which likely accounts for the greater production of systemic anti-GFP antibodies.…”

“…Experiments to evaluate the transduction efficacy, pattern, durability, and cell-type specificity of suprachoroidal AAV8 injections in rhesus macaques using transscleral microneedles have been previously described. 21 Briefly, we identified 5 animals between age 4-10 years with no pre-existing NAbs against AAV8, and injected both eyes with NHP-grade AAV8 that expresses enhanced GFP under a cytomegalovirus (CMV) promoter at 7 x 10 11 vg/eye (low dose) or 7 x 10 12 vg/eye (high dose), using either a 700-m long 30-gauge microneedle (Clearside Biomedical, Alpharetta, GA, USA) for suprachoroidal or transscleral subretinal injection, or a 0.5-inch-long 30-gauge conventional needle for intravitreal injection (Supplementary Table 1). Of these, two animals received suprachoroidal AAV8 in both eyes (Rhesus with x 10 11 vg/eye and Rhesus 02 with 7 x 10 vg/eye), two animals (Rhesus and 04) received suprachoroidal injection of AAV8 in one eye (7 x 10 12 vg/eye) and subretinal delivery of AAV8 in the contralateral eye (7 x 10 12 vg/eye), and the last animal (Rhesus 05) received intravitreal injection of AAV8 in both eyes (7 x 10 12 vg/eye).…”

“…We previously found that suprachoroidal AAV8 injection elicited greater local infiltration of inflammatory cells than transscleral subretinal or intravitreal injections at 1 month postinjection. 21 In this study, we further characterize the local inflammation using immunohistochemistry at 2 and 3 months after suprachoroidal AAV8 delivery ( Figure 2). GFP transgene expression was detectable in both RPE and scleral tissues at 1 month, but only persisted in the sclera at months 2 and 3, appearing mostly in spindle-shaped cells that resemble scleral fibroblasts.…”

“…13,20 We and others have recently described a novel mode of ocular gene delivery by injecting AAV into the suprachoroidal space, which is located between the scleral wall and the choroidal vasculature of the eye. 21,22 Although this potential space is barely detectable under physiologic conditions, 23,24 suprachoroidal injection of compounds using transscleral microneedles expands the suprachoroidal space as seen on in vivo imaging, 25,26 enabling targeted drug delivery to retinal and choroidal tissues while minimizing adverse effects on anterior segment structures. [27][28][29][30][31] Suprachoroidal injection of a triamcinolone acetonide suspension using these microneedles has been effective in treating macular edema from noninfectious uveitis in human clinical trials.…”

Suprachoroidal Delivery of Viral and Nonviral Gene Therapy for Retinal Diseases

“…More recently, microneedle-based suprachoroidal gene delivery was investigated in NHPs. 31 In this study, Yiu et al compared GFP expression among suprachoroidal, subretinal, and intravitreal gene delivery of AAV8 containing an expression cassette encoded for enhanced GFP under a ubiquitous CMV promoter, woodchuck hepatitis virus post-transcriptional regulatory element, and bovine growth hormone polyadenylation signal. Suprachoroidal injection of AAV8-eGFP resulted in widespread peripheral and circumferential transgene expression in RPE, while subretinal AAV8 produced focal transduction in the RPE, PRs, and some ganglion cells, and intravitreal AAV8 resulted in GFP expression in cells, possibly astrocytes, or Müller glia, in peripapillary region.…”