The future of retinal gene therapy: evolving from subretinal to intravitreal vector delivery

Abstract

Inherited retinal degenerations are a leading and untreatbale cause of blindness, and as such they are targets for gene therapy. Numerous gene therapy treatments have progressed from laboratory research to clinical trails, and a pioneering gene therapy received the first ever FDA approval for treating patients. However, currently retinal gene therapy mostly involves subretinal injection of the therapeutic agent, which treats a limited area, entails retinal detachment and other potential complications, and requires general anesthesia with consequent risks, costs and prolonged recovery. Therefore there is great impetus to develop safer, less invasive and cheapter methods of gene delivery. A promising method is intravitreal injection, that does not cause retinal detachment, can lead to pan-retinal transduction and can be performed under local anesthesia in out-patient clinics. Intravitreally-injected vectors face several obstacles. First, the vector is diluted by the vitreous and has to overcome a long diffusion distance to the target cells. Second, the vector is exposed to the host's immune response, risking neutralization by pre-existing antibodies and triggering a stronger immune response to the injection. Third, the vector has to cross the inner limiting membrane which is both a physical and a biological barrier as it contains binding sites that could cause the vector's sequestration. Finally, in the target cell the vector is prone to proteasome degradation before delivering the transgene to the nucleus. Strategies to overcome these obstacles include modifications of the viral capsid, through rational design or directed evolution, which allow resistance to the immune system, enhancement of penetration through the inner limiting membrane or reduced degradation by intracellular proteasomes. Furthermore, physical and chemical manipulations of the inner limiting membrane and vitreous aim to improve vector penetration. Finally, compact non-viral vectors that can overcome the immunological, physical and anatomical and barriers have been developed. This paper reviews ongoing efforts to develop novel, safe and efficacious methods for intravitreal delivery of therapeutic genes for inherited retinal degenerations. To date, the most promising results are achieved in rodents with robust, pan-retinal transduction following intravitreal delivery. Trials in larger animal models demonstrate transduction mostly of inner retinal layers. Despite ongoing efforts, currently no intravitreally-injected vector has demonstrated outer retinal transduction efficacy comparable to that of subretinal delivery. Further work is warranted to test promising new viral and non-viral vectors on large animal models of inherited retinal degenerations. Positive results will pave the way to development of the next generation of treatments for inherited retinal degeneration.

Keywords: adeno-associated virus; animal model; blindness; gene therapy; inner limiting membrane; photoreceptors; retina; retinitis pigmentosa; vitreous.

Figure 1

Schematic representation of subretinal and intravitreal vector injection. Subretinal delivery (left panel) results in s a formation of a bleb of fluid containing the vector between the photoreceptor layer and the retinal pigment epithelium (blue bubble). In intravitreal delivery (right panel), the therapeutic agent is delivered into the vitreous body. Reprinted with permission from Ochakovski et al. (2017). BM: Bruch’s membrane; CC: choriocappilaris; GCL: ganglion cell layer; ILM: inner limiting membrane; INL: inner nuclear layer; IPL: inner plexiform layer; NFL: nerve fiber layer; ONL: outer nuclear layer; OPL: outer plexiform layer; RPE: retinal pigment epithelium.

Figure 2

Barriers to intravitreal delivery. (A) Viral vectors carrying a therapeutic gene for treating inherited retinal degenerations are injected intravitreally. The vector is diluted in the vitreous body, and a small viral load reaches the ILM. Furthermore, in the vitreous the vector is exposed to the host’s immune system, making it vulnerable to neutralization by pre-existing anti-AAV antibodies originating from previous exposure to wild type AAV serotypes, as well as prone to trigger a stronger immune response to the injection. (B) Viral vectors are accumulating along the ILM (which has been manually thickened in this slide). The ILM poses a physical as well as a biological barrier, as it contains AAV binding glycoproteins. Following diffusion through the ILM, the vectors must penetrate a thick and complex extracellular matrix in order to reach target cells in the outer retina, leading to further dilution. (C) A viral vector that reaches the target cell (a photoreceptor in this example) is prone to being marked for ubiquitination and proteasome degradation. Therefore, of all the injected vectors, only one vector successfully delivered the therapeutic gene into the nucleus. Histological slide of panel A courtesy of Richard R Dubielzig. Sourced from the authors’ laboratory. AAV: Adeno-associated virus; GCL: ganglion cell layer; ILM: inner limiting membrane; INL: inner nuclear layer; IPL: inner plexiform layer; NFL: nerve fiber layer; ONL: outer nuclear layer; OPL: outer plexiform layer; PL: photoreceptor layer; RPE: retinal pigment epithelium.