Safety and Efficacy of AAV5 Vectors Expressing Human or Canine CNGB3 in CNGB3-Mutant Dogs

Abstract

Achromatopsia is an inherited retinal disorder of cone photoreceptors characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. Approximately 50% of cases are caused by mutations in the cone photoreceptor-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene. Studies in CNGB3-mutant dogs showed that subretinal injection of an AAV vector expressing human CNGB3, which has 76% amino acid identity with canine CNGB3, driven by a 2.1 kb human red cone opsin promoter (PR2.1) and packaged in AAV5 capsids (AAV5-PR2.1-hCNGB3) rescued cone photoreceptor function, but at high doses was associated with an inflammatory response (focal chorioretinitis) consistent with immune-mediated toxicity. AAV vectors containing the PR2.1 promoter packaged in AAV5 capsids and expressing either the native canine CNGB3 (AAV5-PR2.1-cCNGB3) or the human CNGB3 (AAV5-PR2.1-hCNGB3) were evaluated at different dose levels in CNGB3-mutant dogs. The vector expressing canine CNGB3 achieved somewhat better rescue of cone function but unexpectedly was associated with a greater degree of retinal toxicity than the vector expressing human CNGB3. Very low-level T-cell immune responses to some AAV or CNGB3 peptides were observed in animals that received the higher vector dose. There was a more than twofold increase in serum neutralizing antibodies to AAV in one of three animals in the low-dose group and in two of three animals in the high-dose group. No serum anti-hCNGB3 antibodies were detected in any animal. The results of this study do not support the hypothesis that the focal chorioretinitis seen with high doses of AAV5-PR2.1-hCNGB3 in the initial studies was due to an immune response to human CNGB3.

Keywords: AAV; achromatopsia; chorioretinitis; gene therapy.

Figure 1.

Representative fundus photographs and histologic images taken 14 weeks after subretinal injection of AAV5-PR2.1-hCNGB3 in phenotypically normal dogs from study 1. The vector dose level (vg/eye) and animal number are indicated above the images. (A and B) Other than the retinotomy scars (arrows), no abnormalities could be observed clinically at the 1.0 × 10¹⁰ and 1.0 × 10¹¹ vg doses. Multifocal chorioretinitis within the treated bleb areas developed at the 1.1 × 10¹² and 1.1 × 10¹³ vg doses (“black foci” in C1, C2, D1, and D2). Focal (* in C2) and diffuse (* in D1 and D2) retinal thinning could be recognized as hyper-reflective (brighter than normal) tapetal reflection. Arrowheads (D2) mark the edge of the original bleb area. Examination of hematoxylin and eosin stained sections confirmed the “black foci” as focal aggregates of inflammatory cells within the subretinal space (* in C3) associated with complete loss of photoreceptor inner and outer segments (arrow in C3). The retinal thinning was mainly caused by a severe loss of the outer nuclear layer and photoreceptors (arrow in D3). Perivascular aggregates of inflammatory cells were also observed (* in D3). ONH, optic nerve head. Bars = 50 μm.

Figure 2.

Representative cone flicker electroretinogram (ERG) responses 6 and 12 weeks after subretinal injection of AAV5-PR2.1-hCNGB3 or AAV5-PR2.1-cCNGB3 in CNGB3-mutant dogs (study 3). Animal M730 received the vector expressing human CNGB3 in the left eye (OS) and the vector expressing canine CNGB3 in the right eye (OD).

Figure 3.

Histologic changes in animals receiving the highest vector dose (5 × 10¹¹ vg/eye) of AAV5-PR2.1-hCNGB3 or AAV5-PR2.1-cCNGB3 in study 3. (A) M728 left eye, AAV5-PR2.1-hCNGB3. A retinotomy lesion (thick white arrow) is characterized by focal disruption of outer and inner nuclear layers with prolapse of photoreceptor nuclei into the subretinal space of locally detached retina. The retinal pigment epithelium is intact. Abrupt transition to a zone of marked outer nuclear layer atrophy accompanied by loss of retinal pigment epithelium is apparent (row of black arrows). (B) M729 right eye, AAV5-PR2.1-cCNGB3. Perivascular aggregates of mononuclear inflammatory cells are evident around inner retinal vessels (white arrow). A few inflammatory cells within the vitreous chamber cling to the inner limiting membrane (black arrows). (C) M730 left eye, AAV5-PR2.1-hCNGB3. Inflammatory cells surrounding a choroidal vessel (white arrow) extend into the subretinal space. There is accompanying retinal detachment, atrophy of inner and outer segments, and retinal pigment epithelial attenuation. (D) Boxed region in (C) reveals an inflammatory infiltrate (lymphocytes and plasma cells, black arrows) consistent with an adaptive immune response. Hematoxylin and eosin, bars = 100 μm (A), 50 μm (B and C), and 10 μm (D).