Recombinant adeno-associated viral vector (rAAV)-mediated expression of RLBP1 (CRALBP) rescues the dark adaptation defect in a mouse model of RLBP1-associated retinal degeneration

Abstract

Purpose: RLBP1-associated retinal degeneration is caused by recessive mutations in the RLBP1 gene that lead to absent or non-functional cellular retinaldehyde binding protein (CRALBP) normally expressed in RPE and Müller cells. From early childhood onwards, eyes of patients dark adapt very slowly (up to 24 hours to fully adapt). An associated photoreceptor degeneration causes legal blindness often by middle age. The purpose of this study is to develop a rAAV that can repair the slow dark adaptation in RLBP1-/- mice.

Methods: Recombinant AAV vectors were designed to express enhanced green fluorescent protein (eGFP) or human CRALBP with promoters derived from the human RLBP1 (2 constructs), RPE65, or VMD2 genes, each of which were packaged into 1-4 separate AAV capsid serotypes or genome conformations. The vectors were purified by CsCl or column chromatography. The expression pattern of the vectors encoding eGFP were evaluated by subretinally injecting 1x10⁹ vector genomes (vg) in 1μl in C57BL/6 mice, and 1x10⁹ or 1x10¹⁰ vg in 100μl in Cynomolgus monkeys, and then examining eGFP expression in flat mounts, cryo sections, or paraffin sections of retinas 3 months later. mRNA levels of the human RLBP1 transgene were measured by qPCR of eye tissues from wild type or RLBP1-/- mice 4 weeks after injection. The rate of dark adaptation was determined by electroretinography.

Results: A promoter derived from the human RLBP1 gene drove expression of eGFP primarily in RPE and Müller cells in mouse and cynomolgus retinas. The same promoter was used to express RLBP1 in mice. Of all promoters tested, one derived from RLBP1 elicited the highest vector-mediated mRNA expression per injected vg in both RPE and neural retina in mice. A single subretinal injection of the vector led to a substantial increase in the rate of dark adaptation in RLBP1-/- mice, with dose-response experiments indicating that the EC50 was between 3x10⁷ and 3x10⁸ vg/eye. The improved dark adaptation persisted for at least 1 year.

Conclusions: The human RLBP1 promoter is superior to promoters from the RPE65 or VMD2 genes in driving the expression of a transgene in the RPE and neural retina. A rAAV vector expressing human CRALBP corrects the slow dark adaptation in RLBP1-/- mice. The vector might be therapeutic for patients with RLBP1-associated retinal degeneration.

Keywords: 538 gene transfer/gene therapy • 696 retinal degenerations: hereditary • 705 retinoids/retinoid binding proteins