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. 2023 Oct 10:31:101128.
doi: 10.1016/j.omtm.2023.101128. eCollection 2023 Dec 14.

Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1KO and CRB1KOCRB2+/- retinal organoids

Nanda Boon  1 , Xuefei Lu  1 , Charlotte A Andriessen  1 , Michaela Orlovà  1 , Peter M J Quinn  1 , Camiel J F Boon  1   2 , Jan Wijnholds  1   3
Affiliations

Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1KO and CRB1KOCRB2+/- retinal organoids

Nanda Boon et al. Mol Ther Methods Clin Dev. .

Abstract

The majority of patients with mutations in CRB1 develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, CRB1KO and CRB1KOCRB2+/- differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane. This phenotype with outer retinal abnormalities is similar to CRB1 patient-derived retinal organoids and Crb1 or Crb2 mutant mouse retinal disease models. The CRB1KO and CRB1KOCRB2+/- retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Müller glial cells, suggesting an essential role for CRB1 in proper localization of neuronal cell types. Adeno-associated viral (AAV) transduction was explored at early and late stages of organoid development. Moreover, AAV-mediated gene augmentation therapy with AAV.hCRB2 improved the outer retinal phenotype in CRB1KO retinal organoids. Altogether, these data provide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies.

Keywords: AAV; CRB1; CRB2; CRISPR-Cas9; Müller glial cells; gene therapy; hiPSC; photoreceptor; retinal degeneration; retinal organoids.

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Conflict of interest statement

The authors declare no competing interests. The LUMC is the holder of patent number PCT/NL2014/050549, which describes the potential clinical use of CRB2. J.W. is listed as co-inventor of this patent and is an employee of the LUMC. Horama (currently known as Coave Therapeutics) partially sponsored this study.

Figures

None
Graphical abstract
Figure 1
Figure 1
CRB1KO and CRB1KOCRB2+/− retinal organoids at DD210 (A–C) Representative brightfield images of the (A) isogenic control and (B) CRB1KO and (C) CRB1KOCRB2+/−retinal organoids at DD210, with a zoom-in of the outer segment-like structures in the boxed areas (A′, B′, C′). (D and E) Representative immunohistochemistry images of DD210 isogenic control and two CRB1KO and CRB1KOCRB2+/− retinal organoids stained with (D) rhodopsin and (E) SOX9 (red) and ISLET1-2 (green). INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 50 μm.
Figure 2
Figure 2
CRB1KO and CRB1KOCRB2+/− retinal organoids show more photoreceptor nuclei above the outer limiting membrane compared with the isogenic control at DD180 and DD210 Representative immunohistochemical images of (A) CRB1 (red) co-stained with MUPP1 (green) and (B) CRB2 (green) at the OLM of DD180 control, CRB1KO, and CRB1KOCRB2+/− retinal organoids. (C) Quantification of the number of photoreceptor nuclei above the OLM and (D) number of photoreceptor nuclei in a row of DD180 control, CRB1KO, and CRB1KOCRB2+/− retinal organoids. (E) Quantification of the number of photoreceptor nuclei above the OLM and (F) number of photoreceptor nuclei in a row of DD210 control, CRB1KO, and CRB1KOCRB2+/− retinal organoids. Each datapoint in the graph represents individual organoids, of which an average has been taken of at least three representative images. The standard error of the mean (SEM) is derived from these averages. Number of individual organoids used for the quantification per condition at DD180: 4.10 n = 14, CRB1KO CL19 n = 7, CL26 n = 5, CL72 n = 9, CRB1KOCRB2+/− CL4 n = 3, CL9 n = 8, CL17 n = 8; and DD210: 4.10 n = 12, CRB1KO CL19 n = 9, CL26 n = 10, CL72 n = 11, CRB1KOCRB2+/− CL4 n = 8, CL17 n = 8 from at least two independent differentiation batches and CRB1KOCRB2+/− CL9 n = 5 from one differentiation batch. INL, inner nuclear layer; ONL, outer nuclear layer; OLM, outer limiting membrane. Scale bar, 50 μm, statistical analysis: generalized linear mixed models with p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).
Figure 3
Figure 3
AAV transduction study of control retinal organoids transduced at DD135 with AAV2.CMV.GFP or AAV5.CMV.GFP (A–C) Representative immunohistochemical images of control retinal organoids transduced with (A) 1 × 1010 gc, (B) 6.6 × 1010 gc, or (C) 10 × 1010 gc AAV2.CMV.GFP or AAV5.CMV.GFP. (D and E) Quantification of the number of GFP-positive cells in the (D) ONL and (E) INL. (F and G) Immunohistochemical images of colocalization of AAV.GFP with photoreceptor marker OTX2 (F) or Müller glial cell marker CRALBP (G). Each datapoint in the graph represents individual organoids, of which an average has been taken of 3–6 representative images. The standard error of the mean (SEM) is derived from these averages. Number of individual organoids used for quantification per condition for AAV2.CMV.GFP: 1 × 1010 gc n = 7, 6.6 × 1010 gc n = 7, 10 × 1010 gc n = 10; and for AAV5.CMV.GFP: 1 × 1010 gc n = 9, 6.6 × 1010gc n = 7, 10 × 1010gc n = 11 from at least two independent differentiations. INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 50 μm, statical analysis: generalized linear mixed models with p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).
Figure 4
Figure 4
AAV-mediated gene therapy on CRB1KO organoids transduced at DD120 shows an improved number of photoreceptor nuclei in a row (A–C) Representative immunohistochemical images of (A) untreated, (B) AAV.hCRB1, or (C) AAV.hCRB2 treated control and CRB1KO retinal organoids at DD120 and analyzed at DD210. Stained with MUPP1 (red) at the OLM. (D–G) Quantification of the number of photoreceptor nuclei in a row (D and E) and above the OLM (F and G) per CRB1KO clone (D and F) or all CRB1KO clones combined (E and G). Each datapoint in the graph represents an individual organoid, of which an average has been taken of at least three representative images. The standard error of the mean (SEM) is derived from these averages. Number of individual organoids used for quantification per condition for untreated: 4.10 n = 10, CRB1KO CL19 n = 7, CL26 n = 7, CL72 n = 5; AAV.hCRB1 treated: 4.10 n = 5, CRB1KO CL19 n = 6, CL26 n = 8, CL72 n = 4 from two independent differentiation batches; and AAV.hCRB2 treated: 4.10 n = 5, CRB1KO CL19 n = 4, CL26 n = 6, CL72 n = 3 from one differentiation batch. INL, inner nuclear layer; ONL, outer nuclear layer; OLM, outer limiting membrane. Scale bar, 50 μm, statistical analysis: generalized linear mixed models with p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).
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