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Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy

Abstract

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.

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Fig. 1: Schematic of CRISPR-Cas9 targeting strategy and immunohistochemical and immunoblot demonstration of complete ablation of Pde6b protein expression in Pde6b knockout animals.
Fig. 2: Loss of Pde6b causes severe thinning of the outer nuclear layer due to extensive photoreceptor cell loss.
Fig. 3: Subretinal delivery using two different serotypes of AAV (AAV1 and AAV5) drives human PDE6B in vivo.
Fig. 4: Gene augmentation using AAV1 delays photoreceptor loss to a larger degree than AAV5.

Data availability

All pertinent data generated and analyzed during this study can be found within the published manuscript and supplementary data file.

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Funding

University of Iowa Institute for Vision Research, University of Iowa, Iowa City, Iowa; Research to Prevent Blindness, New York City, New York; Carver cGMP Operational Endowment for Gene Therapy, University of Iowa, Iowa City, Iowa; National Institutes of Health Grant (NIH; Bethesda, MD, USA): P30 EY025580.

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ICH and LAW conceived of, designed, performed experiments, analyzed data and wrote the manuscript. ICH, LAW, DO, MJL, BEH and KMS acquired samples and helped perform experiments. EMS and RFM provided funding support, edited and approved the final manuscript. BAT conceived the study, provided funding support, as well as edited and approved the final manuscript.

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Correspondence to Budd A. Tucker.

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The authors declare no competing interests.

Ethical approval

As also stated in the methods section, all rat experiments were conducted with the approval of the University of Iowa Animal Care and Use Committee (Animal welfare assurance #1031317) and were consistent with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

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Han, I.C., Wiley, L.A., Ochoa, D. et al. Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy. Gene Ther (2022). https://doi.org/10.1038/s41434-022-00365-y

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