AAV-mediated gene-replacement therapy restores viability of BCD patient iPSC derived RPE cells and vision of Cyp4v3 knockout mice

Abstract

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal degenerative disease characterized by yellow-white crystal deposits in the posterior pole, degeneration of the retinal pigment epithelium (RPE), and sclerosis of the choroid. Mutations in the cytochrome P450 4 V2 gene (CYP4V2) cause BCD, which is associated with lipid metabolic disruption. The use of gene replacement therapy in BCD has been hampered by the lack of disease models. To advance CYP4V2 gene replacement therapy, we generated BCD patient-specific induced pluripotent stem cell (iPSC)-RPE cells and Cyp4v3 knockout (KO) mice as disease models and AAV2/8-CAG-CYP4V2 as treatment vectors. We demonstrated that after AAV-mediated CYP4V2 gene replacement therapy BCD-iPSC-RPE cells presented restored cell survival and reduced lipid droplets accumulation; restoration of vision in Cyp4v3 KO mice was revealed by elevated electroretinogram amplitude and ameliorated RPE degeneration. These results suggest that AAV-mediated gene replacement therapy in BCD patients is a promising strategy.

In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment

“…For the corresponding donor plasmid, the CMV promotor was also replaced by the EFS promotor, the constructed vector is pX601-EFS-Puro-P2A-mCherry-donor3. Construct the pAV-CAG-CYP4V3(E1-6)-CYP4V2(E7-11) plasmid containing desired transcription sequence based on pAV-CAG-CYP4V2 from previous study 20 . The fragment of CYP4V2(E1-6) was replaced by CYP4V3(E1-6) (NM_133969.3).…”

“…For Western blot, cells or mouse retinas extracted from sacrificed mouse following removal of the anterior segments and lens were homogenized in radio immunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor cocktail in a homogenizer, following the same steps as described 20 . The targeted bands were detected using enhanced chemiluminescence (ECL; Millipore, MA, USA) exposure reagent and collected using a ChemiDoc™ MP imaging system (Tanon Science & Technology Co., Shanghai, China).…”

“…Recently, several teams including ourselves have reported encouraging results on CYP4V2 gene replacement therapy in BCD-iPSC-RPE cells and Cyp4v3 KO mice 18 – 20 . Our team has developed a CYP4V2 gene replacement therapy drug ZVS101e 21 , and two Investigator-Initiate Clinical Trials (IIT; NCT04722107 and NCT05714904) have been launched since June 2021.…”

Gene replacement therapy in Bietti crystalline corneoretinal dystrophy: an open-label, single-arm, exploratory trial

“… 8 The CYP4V2 protein is produced in multiple body tissues but is particularly abundant in the RPE. 9 , 10 CYP4V2 is an omega 3-polyunsaturated medium-chain fatty acid hydroxylase that hydrolyzes docosahexaenoic and eicosapentaenoic acid in the eye. 9 The photoreceptor outer segment contains large amounts of lipids that can be esterified to form docosahexaenoic acid, eicosapentaenoic acid, and other fatty acids.…”

“… 12 – 15 In particular, the recombinant AAV2/8–human CYP4V2 (rAAV-h CYP4V2 ) vector, which produces the functional wild-type human CYP4V2 protein (patent no. CN113106124B), has been found to be efficient in an induced pluripotent stem cells-derived RPE from a patient with BCD as well as in a Cyp4v3- knockout mouse model, 14 providing the basis for a clinical trial for this gene therapy.…”