Noninvasive gene delivery to foveal cones for vision restoration

Abstract

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.

Keywords: Gene therapy; Genetic diseases; Ophthalmology; Surgery; Therapeutics.

Figure 1. Adeno-associated viral (AAV) vector administration route defines retinal transduction patterns with mCAR, PR2.1, and PR1.7 promoters.

(A–C) Representative retinal cross sections of WT mouse retinas (n = 6 eyes per condition) 3 weeks after subretinal injection of AAV2-7m8-mCAR-GFP (A), AAV2-7m8-PR2.1-GFP (B), and AAV2-7m8-PR1.7-GFP (C). (D–F) Representative retinal cross sections 2 months after intravitreal injection (n = 6 eyes per condition) of AAV2-7m8-mCAR-GFP (D), AAV2-7m8-PR2.1-GFP (E), and AAV2-7m8-PR1.7-GFP (F). Scale bar: 50 μm in A–F. (G) GFP expression in rd10 retina (n = 4 eyes) 2 months after intravitreal injection of AAV2-7m8-PR1.7-GFP. Transduced cone cell bodies remaining after degeneration express GFP (cyan). Cone arrestin is shown in magenta, and DAPI is shown in blue. Native GFP expression is shown in cyan, and arrows indicate cells where cone arrestin is colocalized with GFP. Scale bar is 50 μm in G. mCAR, mouse cone arrestin promoter; PR1.7 and PR2.1, promoters of 1.7 and 2.1 kilobases in length, respectively, based on the human red opsin gene enhancer and promoter sequences.

Figure 2. Model for the regulation of transgene expression under the control of PR2.1 and PR1.7 synthetic promoters and mouse cone arrestin (mCAR) promoter.

(A) Red opsin gene is located on the X chromosome. It has its own proximal promoter and shares its enhancer sequence with green opsin gene. Chromosomal loops between the enhancer and the red opsin proximal promoter provide cell-type specificity of gene expression. PPred, proximal promoter of red opsin gene; LCR, locus control region. (B) Schematic representation of PR2.1 and PR1.7 promoter constructs. Interaction with inhibitory transcription factors such as COUP-TFI (chicken ovalbumin upstream promoter-transcription factor), that binds the 337bp region specific to PR2.1 might explain low expression levels obtained with PR2.1 compared with PR1.7 in macaque cones subretinally (18). On the other hand, activator TFs such as CEBPB (CCAAT/enhancer binding protein β) and GTF2I (general transcription factor II-I) that are not specific to cones likely lead to off-target expression in other retinal cells when injected into the vitreous. ITRs, inverted terminal repeats. (C) Structure of cone arrestin 3 genomic locus region. Transcription starting sites (TSS) of mouse arrestin 3 (mArr3) gene and mouse pyrimidinergic receptor P2Y4 (mP2ry4) gene are separated by 10.5 kilobases. The short 521-bp mCAR promoter used in this study is shown in blue and the supposed regulatory region referred to as “Reg” in magenta. (D) Structure of the 521-bp mCAR promoter portion used in this study. This sequence contains CRX-binding sites (CBS) and SP (Specificity Protein) binding sites. It also contains 1 TATA and 1 TATA-like box. (E) Interactome network of several transcription factors that bind cone arrestin genomic promoter analyzed using the STRING tool. CRX (cone-rod homeobox protein), SP, RARA (retinoic acid receptor α), RXRA (retinoid X receptor α), and THRB (thyroid hormone receptor β 2). (F) NR-MED1 transcription regulator complex confers gene expression specificity. MED1 (mediator complex subunit 1) is a transcription activator when associated to nuclear receptors (NRs). RARA, RXRA, and THRB are NRs. Several NR binding sites for RARA, RXRA, and THRB were found in the Reg region. AAV, adeno-associated virus; mCAR, mouse cone arrestin promoter; PR1.7 and PR2.1, promoters of 1.7 and 2.1 kilobases in length, respectively, based on the human red opsin gene enhancer and promoter sequences.

Figure 3. Foveal cone transduction with PR1.7 promoter versus cytomegalovirus (CMV) promoter 2–3 months after intravitreal delivery of AAV2-7m8 in nonhuman primates.

Representative eye fundus images after intravitreal injection of AAV2-7m8-CMV-GFP (n = 2 eyes) (A and B) or AAV2-7m8-PR1.7-GFP (n = 2 eyes) (C and D) at 1 × 10¹¹ viral particles per eye. B and D are inset of images shown in A and C. Scale bars: 200 μm. Confocal images of the maculas mounted with the ganglion cell layer facing upward using CMV (E) and PR1.7 (F) promoters. Scale bars: 500 μm. (G and H) Zoomed images of the fovea with CMV (G) and with PR1.7 (H). Scale bars: 100 μm in G and H. (I–K) Retinal cryosections at the level of the fovea. (I) DAPI staining at the level of the fovea. Asterisk represents foveal pit. GFP expression under the control of CMV (J) or PR1.7 (K) promoters. Scale bar: 50 μm in I, J, and K. (L–N) Confocal image projection of the whole foveal flatmount showing nuclei (L) and GFP expression in cones (M). Scale bar: 100μm. (N) Zoom into 3-D–reconstructed fovea seen in M with close-up to the cell bodies (facing upward). Scale bar: 10μm. AAV, adeno-associated virus; PR1.7, a promoter of 1.7 kilobases in length, based on the human red opsin gene enhancer and promoter sequences.

Figure 4. Optical coherence tomography (OCT) follow-up of AAV2-7m8-CMV-GFP– and PR1.7-GFP–treated eyes.

(A) Foveal OCT images of CMV-treated eyes (n = 2). (B) Foveal OCT images of PR1.7-treated eyes (n = 2). D0, day of injection, predose; M1.5, -2, -3, month 1.5, 2, or 3 after dose; AAV, adeno-associated virus; CMV, cytomegalovirus promoter; PR1.7, promoter of 1.7 kilobases in length, based on the human red opsin gene enhancer and promoter sequences.

Figure 5. Optogenetic activation of foveal cones using AAV2-7m8-PR1.7-Jaws-GFP.

(A) Infrared eye fundus image and (B) optical coherence tomography (OCT) image of the eye injected intravitreally with AAV2-7m8-PR1.7-Jaws-GFP (n = 1, 1 × 10¹¹ vg and n = 1, 1 × 10¹⁰ vg). (C) Eye fundus fluorescence image 2 months after injection shows Jaws-GFP expression in the fovea. Inset magnification (B and C): ×1.5. (D) Half foveal flatmount showing efficient and specific foveal transduction using AAV2-7m8-PR1.7-Jaws-GFP. Scale bar: 50 μm. Arrow, foveola; red rectangle, close-up to the foveola shown in retinal sections in E; scale bar: 20 μm. (F–K) Characteristics of the cone photoreceptor light responses triggered by optogenetic stimulation of Jaws in living macaque retinas (n = 4 cells). (F) Superimposed infrared and epifluorescence images showing strong Jaws-GFP fluorescence in the foveal cones of patched explants. (G) Infrared image of the same tissue. Patch electrode (black dotted line) is shown in contact with a Jaws-GFP⁺ cone cell body highlighted in cyan. ONL, outer nuclear layer; IS, inner segments; OS, outer segments. (H and I) Whole-cell patch clamp recordings of Jaws-GFP–expressing macaque cones. Jaws-induced photocurrents as a function of light intensity. Orange light stimulation ranged from 1 × 10¹⁴ to 3 × 10¹⁷ photons cm^–2·s^–1. (J) Jaws-induced photocurrents as a function of stimulation wavelength in intravitreally injected macaque eye. Stimuli were applied from 400–650 nm, separated by 25-nm steps, at an intensity equal to 8 × 10¹⁶ photons cm^–2·s^–1. Maximal responses were obtained at 575 nm. (K) Jaws-GFP–expressing cones recorded in current-clamp configuration in current zero mode (with their resting membrane potential indicated in gray), displaying light-elicited hyperpolarizations followed by short depolarizations. AAV, adeno-associated virus; PR1.7, promoter of 1.7 kilobases in length, based on the human red opsin gene enhancer and promoter sequences.

Figure 6. AAV9-7m8 transduces the fovea via delivery in a distal bleb and provides robust optogenetic light responses with PR1.7-Jaws.

(A) Eye fundus infrared image and (B) optical coherence tomography (OCT) image immediately after subretinal delivery of AAV9-7m8 in peripheral retina. (C) Eye fundus fluorescence image 1 month after injection shows strong Jaws-GFP expression within the subretinal bleb and away from the injection site, including the fovea. Inset magnification: ×1.5. (D–F) Foveal flatmount shows highly efficient and specific foveal transduction using subretinal AAV9-7m8-PR1.7-Jaws-GFP. Scale bar: 50 μm. (G–L) Characteristics of the light responses triggered by optogenetic stimulation of Jaws. (G) Lateral view of Jaws-expressing cones in living tissue using 2-photon imaging. (H and I) Whole-cell patch clamp recordings of Jaws-GFP⁺ macaque cones. Jaws-induced photocurrents as a function of light intensity. Stimuli were applied from 1 × 10¹⁴ to 3 × 10¹⁷ photons cm^-2 s^-1 (n = 9 cells from 2 retinas of 2 animals). (J) Jaws-GFP⁺ cones recorded in current-clamp configuration in current zero mode (with resting membrane potential indicated in gray), displaying light-elicited hyperpolarizations followed by short depolarizations. (K) Jaws-induced photocurrents as a function of stimulation wavelength in subretinally injected macaque eye. Stimuli were applied from 400–650 nm, separated by 25-nm steps, at an intensity equal to 8 × 10¹⁶ photons cm^–2·s^–1. Maximal responses were obtained at 575 nm (asterisk). (L) Characterization of temporal properties. Modulation of Jaws-induced membrane photocurrents at increasing stimulation frequencies in Jaws-expressing macaque cones, from 2–30 Hz, at 8 × 10¹⁶ photons cm^–2·s^–1. AAV, adeno-associated virus; PR1.7, promoter of 1.7 kilobases in length, based on the human red opsin gene enhancer and promoter sequences. IR, infrared.

Figure 7. Performance of AAV2-7m8-PR1.7 vector–promoter combination in human cones.

(A–C) GFP expression in human induced pluripotent stem cell–derived (iPSC-derived) retinal organoids (n = 10 organoids) infected with AAV2-7m8-PR1.7-GFP. (A) Brighfield, (B) epifluorescence, and (C) confocal images of 43-day-old whole mount organoids infected with AAV2.7m8-PR1.7-GFP at day 28 with a dose of 5 × 10¹⁰ vg/organoid. Scale bar: 200 μm in A and B, and 250 μm in C. Outline in C represents the edges of the organoids (D–F) Retinal organoid cryosections for visualization of GFP expression (cyan). Transduced cones are visualized by superimposition of GFP (cyan) and human cone arrestin (hCAR) immunostaining (magenta). Scale bar: 20 μm in D–F. Arrows represent colocalization of GFP and hCAR stainings. (G–I) Efficient and specific transduction of human cones in postmortem retinal explants. (G) Postmortem human retinal explant placed in culture. Dashed circle shows the approximate area where 1 × 10¹⁰ viral particles were deposited onto the explant (n = 2 explants from 2 eyes of a single donor). (H) Close-up of the transduced area showing high-level GFP fluorescence in region of the explant in contact with the vector. Scale bar: 100 μm. (I) GFP expression (cyan) is restricted to the photoreceptor layer as shown by DAPI (blue) staining. (J) GFP is expressed in cones as shown by colocalization of GFP staining of cone markers, namely M/L opsin. Scale bar: 50 μm in I–J. Arrows represent colocalization of GFP and M/L opsin stainings. AAV, adeno-associated virus; vg, viral genome; PR1.7, promoter of 1.7 kilobases in length, based on the human red opsin gene enhancer and promoter sequences.

Figure 8. Vector delivery strategies to meet therapeutic gene expression requirements.

(A) Central subretinal injection is the most risky and can be associated to adverse effects in the macula. (B) Peripheral subretinal injection using classical vectors does not reach the fovea; however, use of AAV9-7m8 is a promising strategy for achromatopsia patients. (C) Intravitreal injection is surgically simpler and the safest administration route to transduce cones of the foveola, the region responsible for high-acuity vision. It is a preferred delivery approach for retinitis pigmentosa patients to benefit from optogenetic therapy. AAV, adeno-associated virus.