Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies

Abstract

Purpose: Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy.

Methods: We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C. Transduction, transgene expression, and biological activity were characterized in human retinal cells in vitro and in NHPs.

Results: R100 demonstrated superior retinal cell transduction in vitro and in vivo compared to AAV2, a commonly used wild-type AAV serotype in retinal gene therapies. Transduction of human retinal pigment epithelial cells in vitro by 4D-150 resulted in dose-dependent transgene expression and corresponding reductions in VEGF-A and VEGF-C. Intravitreal administration of 4D-150 to NHPs was well tolerated and led to robust retinal expression of both transgenes. In a primate model of laser-induced choroidal neovascularization, 4D-150 completely prevented clinically relevant angiogenic lesions at all tested doses.

Conclusions: These findings support further development of 4D-150. Clinical trials are underway to establish the safety and efficacy of 4D-150 in individuals with wet AMD and DME.

Figure 1.

Directed evolution of AAV capsid in primates led to the discovery of a dominant capsid variant (R100). (A) Schematic representation of directed evolution. A plasmid library comprising synthetic variant AAV capsid sequences is created from the cap genes of several wild-type AAV serotypes using various DNA mutation techniques. The library is packaged in HEK293T cells to produce viral particles comprising a synthetic capsid shell surrounding the viral capsid genome. The viral library is then purified and subjected to in vivo selective pressures. Successful viruses are amplified, recovered, and enriched through repeated rounds of selection. (B) Frequency of the top two capsid variants in the directed evolution round six sequencing analysis. (C) Frequency of the R100 variant motif found in separate retinal cell layers in directed evolution rounds three to six. Error bars indicate 95% confidence intervals. (D) Representative 3D model of R100. The AAV2-based variant contains an insertion of 10 amino acids (purple) at amino acid position 588. AAV, adeno-associated virus.

Figure 2.

In vitro characterization of R100 in human RPE cells compared to AAV2. (A, B) Representative images and quantitation of EGFP transgene expression (green) 7 days post infection in A hESC-derived and B iPSC-derived RPE cells expressing ZO-1 (red) transduced with R100 or AAV2. Line graphs show EGFP+ cells in the PMEL17+ RPE cell population, quantified by flow cytometry. (C) EGFP expression kinetics following transduction by R100 or AAV2 in iPSC-derived RPE cells during the first 7 days post infection. In C, nuclei were counterstained with DAPI (blue). All images are from cultures transduced at an MOI of 5000 except for C, which depicts cultures transduced at an MOI of 2500. All quantitative measurements were carried out in n = 3 replicates; data are mean ± SD. *P < 0.05 compared to AAV2 by two-tailed t test. AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; MOI, multiplicity of infection; RGC, retinal ganglion cell; RPE, retinal pigment epithelium.

Figure 3.

R100-mediated retinal transgene expression after IVT injection. All data were acquired from retinas in NHPs following administration of R100.CAG-EGFP or vehicle control. Representative fundus fluorescence images from (A) an eye injected with vehicle control and an eye injected with R100.CAG-EGFP 1 × 10¹² vg/eye. (B) Representative heat map visualization of IHC scoring (NHP #304, OD). EGFP expression in the RPE layer (left), PR layer (middle), and RGC layer (right) was quantified for each assessed visual field and depicted as shades of blue corresponding to the estimated percentage of EGFP+ cells within each retinal layer. Each section was assigned a score of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%) based on the estimated proportion of cells expressing EGFP. (C) Assessment of EGFP expression in NHP eye sections by IHC 8 weeks after administration of R100.CAG-EGFP 3 × 10¹¹ vg/eye or 1 × 10¹² vg/eye. (D) Representative image of transduced cells in the central retina showing robust EGFP expression in foveal cone PRs (white) and RGCs. (E, F) Representative images demonstrating robust EGFP expression in peripheral PRs E and co-localized with opsin (red) F. (G–I) Representative images of EGFP expression in RPE G, the outer segments of PR H, and the axons of RGCs I. EGFP expression (green) is also detected by an anti-GFP antibody (red) in all images except F. Nuclei were counterstained with DAPI (blue) except in G. EGFP, enhanced green fluorescent protein; IHC, immunohistochemistry; IVT, intravitreal injection; NHP, nonhuman primate; PR, photoreceptor; RGC, retinal ganglion cell; RPE, retinal pigment epithelium.

Figure 4.

Widespread transduction of the NHP retina after IVT administration of R100 compared to AAV2. Data were acquired from retinas in African green monkeys 4 to 10 weeks post administration of 2 × 10¹¹ vg/eye of R100.CMV-EGFP or AAV2.CMV-EGFP (N = 3 animals and N = 4 eyes per group). All animals were seronegative for pre-existing neutralizing antibodies to the R100 and AAV2 capsids. The lower exposure setting in the R100 images was necessary to compensate for the strength of the GFP signal; formal quantification of the GFP signal was not feasible. AAV, adeno-associated virus; IVT, intravitreal injection; NHP, nonhuman primate.

Figure 5.

Structure–function analysis demonstrating a disruption of the heparan sulfate binding domain in R100 through a 10-amino-acid peptide insertion in the GH loop and an increase in retinal cell binding. Molecular models of the GH loop within the VP3 common region of (A) AAV2 and (B) R100. The R100 peptide sequence (magenta) is constrained by the location of amino acids R585 and R588 (blue) within the intact capsid. (C) AAV2 and R100 vectors bound to and eluted from a heparin affinity column. R100 capsid elutes at a lower conductivity compared to AAV2, demonstrating a decrease in heparin affinity. (D) AAV vectors were used to transduce Hap1 and AAVR KO Hap1 cell lines. All vectors demonstrate a cell surface receptor dependence on AAVR. N = 3 replicates each. *P < 0.05 by paired t test between Hap1 and AAVR KO within each serotype. (E) Representative images of AAV2 and R100 binding to human iPSC-derived RPE (MOI = 2500), visualized using an anti-AAV2 capsid antibody (red). (F) The number of AAV2 and R100 viral genomes bound to human iPSC-derived RPE was quantified across a range of MOIs. Error bars indicate standard deviation. *P < 0.05 by two-tailed t test. AAV, adeno-associated virus; AAVR, AAV receptor; iPSC, induced pluripotent stem cell; KO, knockout; MOI, multiplicity of infection; RPE, retinal pigment epithelium.

Figure 6.

The 4D-150 dual-transgene expression and function in vitro. (A) Schema of 4D-150, a dual-transgene anti-angiogenic AAV gene therapy product. (B) Analysis of VEGF-A protein neutralization in the medium of human RPE transduced with 4D-150 or control R100.CAG-GFP vector, data are mean ± SD, *P < 0.05, ****P < 0.0001 by 2-way ANOVA with Tukey's multiple comparison post hoc test. (C) Expression of mature anti-VEGF-C miRNA measured by quantitative reverse transcription PCR (RT-qPCR) from transduced human RPE (MOI = 1000) in response to 4D-150 treatment, data are mean ± SD, **P < 0.005 by unpaired t test. (D) RT-qPCR analysis of VEGF-C transcript levels in transduced RPE (MOI = 1000), normalized to Ribosomal Protein L32 as the housekeeping control. Mean ±SD. (E) Analysis of secreted VEGF-C protein from transduced RPE (MOI = 1000). BLQ values are represented as “0”, data are mean ± SD. ****P < 0.0001 by unpaired t test. Analysis of (F) proliferation and (G) migration of HUVEC cultures in a Transwell system after electroporation with the respective plasmid construct (N = 3, data are mean ± SD. **P < 0.01 compared to shock (electroporation control), *P < 0.05 compared to shock, ^††P < 0.01 compared to CAG-GFP by 1-way ANOVA with Tukey's multiple comparison post hoc test. AFLB, aflibercept; BLQ, below the limit of quantification; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; ITR, inverted terminal repeats; MOI, multiplicity of infection; RLU, relative luciferase units; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor.

Figure 7.

Aflibercept protein expression and laser-induced CNV lesions in primates following IVT administration of 4D-150 or control. (A) Secreted free aflibercept protein expression in the aqueous humor by treatment group 21 days post IVT administration (n = 14 eyes per group) and prior to laser-induced CNV, data are mean ± SD. ****P < 0.0001 by 1-way ANOVA with Bonferroni's multiple comparisons. (B) Percentage of eyes with grade IV lesions 2 weeks post laser per treatment group. **P < 0.005, *P < 0.01 compared to vehicle by Fisher's Exact Prob test. (C) Reduced CNV complex area with 4D-150 pretreatment at 4 weeks post laser. Data are mean ± SEM. CNV complex areas of the principal axis of each lesion were calculated for each treatment group. Pretreatment with all doses of 4D-150 resulted in significantly smaller mean CNV complex areas compared to vehicle. **P < 0.005 by 2-way ANOVA followed by Tukey-Kramer HSD. AFLB, aflibercept; CNV, choroidal neovascularization; IVT, intravitreal.