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.

A Single-Cell Temporal Atlas of Mouse Nasal Embryonic Development

“…smFISH confirmed OE-specific expression of Cyp4v3 (Fig. 2F), a member of the cytochrome P450 family, which participated in lipid metabolism and steroid hormone biosynthesis (Jia et al, 2023). Cldn9, encoding a tight junction protein, may contribute to olfactory epithelium barrier integrity (Nakano et al, 2009;Bird et al, 2010).…”

A Single-Cell Temporal Atlas of Mouse Nasal Embryonic Development

“…smFISH confirmed OE-specific expression of Cyp4v3 (Fig. 2F), a member of the cytochrome P450 family, which participated in lipid metabolism and steroid hormone biosynthesis (Jia et al, 2023). Cldn9, encoding a tight junction protein, may contribute to olfactory epithelium barrier integrity (Nakano et al, 2009;Bird et al, 2010).…”

Gene therapy induces pigmentation in induced pluripotent stem cell-derived retinal pigment epithelium of OCA1A patients and albino rat eye

“…Multiple studies published in recent years signify the utility of iPSC-derived RPE as an in vitro model to perform proof-of-principle studies for rescuing defects observed in the patient-specific RPE. 21 , 22 , 23 , 24 , 25 , 26 A key strength of our study is the use of the same AAV construct for both human iRPE and a rodent model, whereas prior studies primarily restricted their constructs to either human iRPE or mouse models only. Moreover, most gene therapy studies rely on mouse models, even though the AAV serotypes employed often fail to transduce human tissues efficiently.…”

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).…”

A Single-Cell Temporal Atlas of Mouse Nasal Embryonic Development

“…smFISH confirmed OE-specific expression of Cyp4v3 (Fig. 2F), a member of the cytochrome P450 family, which participated in lipid metabolism and steroid hormone biosynthesis (Jia et al, 2023). Cldn9, encoding a tight junction protein, may contribute to olfactory epithelium barrier integrity (Nakano et al, 2009;Bird et al, 2010).…”

Gene therapy induces pigmentation in induced pluripotent stem cell-derived retinal pigment epithelium of OCA1A patients and albino rat eye

“…Multiple studies published in recent years signify the utility of iPSC-derived RPE as an in vitro model to perform proof-of-principle studies for rescuing defects observed in the patient-specific RPE. 21 , 22 , 23 , 24 , 25 , 26 A key strength of our study is the use of the same AAV construct for both human iRPE and a rodent model, whereas prior studies primarily restricted their constructs to either human iRPE or mouse models only. Moreover, most gene therapy studies rely on mouse models, even though the AAV serotypes employed often fail to transduce human tissues efficiently.…”

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).…”

A Single-Cell Temporal Atlas of Mouse Nasal Embryonic Development

“…smFISH confirmed OE-specific expression of Cyp4v3 (Fig. 2F), a member of the cytochrome P450 family, which participated in lipid metabolism and steroid hormone biosynthesis (Jia et al, 2023). Cldn9, encoding a tight junction protein, may contribute to olfactory epithelium barrier integrity (Nakano et al, 2009;Bird et al, 2010).…”

Gene therapy induces pigmentation in induced pluripotent stem cell-derived retinal pigment epithelium of OCA1A patients and albino rat eye

“…Multiple studies published in recent years signify the utility of iPSC-derived RPE as an in vitro model to perform proof-of-principle studies for rescuing defects observed in the patient-specific RPE. 21 , 22 , 23 , 24 , 25 , 26 A key strength of our study is the use of the same AAV construct for both human iRPE and a rodent model, whereas prior studies primarily restricted their constructs to either human iRPE or mouse models only. Moreover, most gene therapy studies rely on mouse models, even though the AAV serotypes employed often fail to transduce human tissues efficiently.…”

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).…”