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. 2018 Sep 4;115(36):E8547-E8556.
doi: 10.1073/pnas.1805055115. Epub 2018 Aug 20.

Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector

Artur V Cideciyan  1 , Raghavi Sudharsan  2 , Valérie L Dufour  2 , Michael T Massengill  3 , Simone Iwabe  2 , Malgorzata Swider  1 , Brianna Lisi  1 , Alexander Sumaroka  1 , Luis Felipe Marinho  2 , Tatyana Appelbaum  2 , Brian Rossmiller  3 , William W Hauswirth  4 , Samuel G Jacobson  1 , Alfred S Lewin  5 , Gustavo D Aguirre  2 , William A Beltran  6
Affiliations

Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector

Artur V Cideciyan et al. Proc Natl Acad Sci U S A. .

Abstract

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.

Keywords: RHO; RNA interference; autosomal dominant retinitis pigmentosa; gene therapy; retinal degeneration.

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Conflict of interest statement

Conflict of interest statement: A.V.C., M.T.M., W.W.H., S.G.J., A.S.L., G.D.A., and W.A.B. are inventors on US Patent Application no. PCT/US2017/020289 and US Provisional Application No. 62/679,585. W.W.H. and the University of Florida have a financial interest in the use of adeno-associated virus therapies and own equity in a company (AGTC, Inc.). The University of Florida and University of Pennsylvania have licensed the gene therapy technology discussed in the work to Ophthotech Corp.

Figures

Fig. 1.
Fig. 1.
shRNA-mediated knockdown of WT, P23H, T17M, and shRNA820-resistant (RHO820) variants of human RHO. HEK293T cells were transfected with a plasmid expressing WT, P23H, T17M, or shRNA820-resistant (RHO820) human RHO with a C-terminal turboGFP tag (RHO-tGFP) and with a rAAV2 plasmid (denoted in lane labels) encoding empty DNA (no shRNA), a control shRNA, shRNA131, shRNA134, or shRNA820. A no-DNA transfection control was also included. (A, C, E, and G) Immunoblots of protein samples isolated from transfected HEK293T cells probed for turboGFP tag (green) and β-tubulin (red) as the loading control. Rho aggr., aggregated form of RHO-GFP; Rho mono., monomeric form of RHO-GFP. (B, D, F, and H) Relative quantification of the monomeric form of RHO-GFP (Upper) and of the monomeric and aggregated forms of RHO-GFP (Lower). The first lane of each Western blot contained the Chameleon Duo Prestained Protein Ladder from Li-Cor. Bars denote the mean value of three technical replicates; error bars denote SEM. ns, not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 2.
Fig. 2.
Suppression of rhodopsin with shRNA820 in WT retinas. (A–C) In vivo imaging results from representative WT eyes 7–8 wk postinjection with scAAV2/5-H1-shRNA820 at 1 × 1011 (B) and 5 × 1011 vg/mL (C) titers compared with uninjected control (A). Shown are OCT scans (Left), normalized IS/OS intensity topography (Center), and ONL thickness topography (Right). Dotted lines indicate injection bleb boundaries. Arrows indicate the location of the OCTs shown on left. (D and E) Normalized IS/OS intensity (D) and ONL thickness (E) sampled within the injected blebs (green symbols, Upper) and uninjected control locations (red symbols, Lower) in 10 eyes injected with a range of titers. Symbols represent group averages (± SD) from 33–95 samples (SI Appendix, Fig. S6). Dashed lines denote the 99th percentile limits of the respective parameters sampled at the same retinal locations in uninjected control eyes. Downward arrows estimate the titers corresponding to the transitions to a detectable effect. (F) Microphotographs of H&E-stained (Upper) and rhodopsin (RHO, green) immunolabeled (Lower) retinal cryosections showing the morphology of the ONL and outer segments (OS) in areas treated with 1 × 1011 to 10 × 1011 vg/mL titer range (Tx) and untreated areas (UnTx) 7–8 wk postinjection. (G) Schematic representation of the retinas of WT dogs treated with 1 × 1011 to 50 × 1011 vg/mL titers showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression 7–8 wk postinjection. Dashed lines indicate bleb boundaries; the blue area indicates the tapetal region. (H) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with the different vector titers. (I) Representative immunoblot and quantification of the levels of endogenous canine RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with the different vector titers. Labels such as N282-OD refer to the animal and the eye; OSasOD indicates the left eye is displayed as the right eye for comparability.
Fig. 3.
Fig. 3.
Suppression of rhodopsin with shRNA820 in RHO-mutant retinas. (A) Schematic representation of the fundus of four RHO-mutant dog eyes injected with scAAV2/5-H1-shRNA820 at 1 × 1011 to 10 × 1011 vg/mL titers showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression at 8–10 wk postinjection. Dashed lines indicate bleb boundaries; the blue area indicates the tapetal region. (B) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with different titers. (C) Representative immunoblot and quantification of the levels of endogenous canine RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of eyes injected with 1 × 1011 to 50 × 1011 vg/mL titers. (D) ONL thickness topography 2 wk post light exposure (8–10 wk postinjection) in four RHO-mutant dog eyes treated with 1× 1011 to 10 × 1011 vg/mL titers. Dotted lines indicate bleb boundaries; dashed lines indicate ONL rescue boundaries. (Insets) Maps of significance showing retinal regions with ONL thickness (Left) and IS/OS intensity (Right) values compared point by point to the 99th percentile CIs of uninjected controls. (E) Microphotographs of H&E-stained (Upper) and rhodopsin (RHO, green)/human cone arrestin (hCA, red) coimmunolabeled (Lower) retinal cryosections showing morphology of the ONL and outer segment (OS) 2 wk post light exposure (8–10 wk postinjection) in areas treated with 1× 1011 to 10 × 1011 vg/mL titer range (Tx) and untreated areas (UnTx) of the eyes shown in D.
Fig. 4.
Fig. 4.
Suppression and replacement of rhodopsin with a single vector prevents retinal degeneration in RHO-mutant retinas. (A and B) ONL thickness topography after injection/before light exposure (post Inj.) and 2 wk post light exposure (post LE) in two RHO-mutant eyes injected with scAAV2/5-hOP-RHO820-H1-shRNA820 at 5 × 1011 vg/mL titer. Dotted lines indicate bleb boundaries; dashed lines indicate ONL rescue boundaries. (Insets) Maps of significance as described in Fig. 3. (C and D) Retinal cryosections coimmunolabeled with rhodopsin (RHO, green)/human cone arrestin (hCA, red) showing morphology of the ONL and outer segment (OS) in treated and untreated areas of the eyes shown in A and B. (E) Schematic representation of the fundus of four RHO-mutant dog eyes injected with a 5 × 1011 vg/mL titer showing the location of neuroretinal punches used for quantification of rhodopsin (RNA and protein) expression. Dashed lines indicate bleb boundaries; the blue area indicates the tapetal region. (F) Quantification of the levels of endogenous canine RHO RNA remaining in the treated retinal area as a percentage of levels measured in the untreated area of injected eyes. (G) Quantification of the levels of exogenous human RHO RNA (RHO820) present in the treated retinal area as a percentage of physiological levels of endogenous canine RHO measured in the untreated area of injected eyes. (H) Representative immunoblot and quantification of the levels of total (endogenous canine and RHO820) RHO protein remaining in the treated retinal area as a percentage of levels measured in the untreated area of injected eyes.
Fig. 5.
Fig. 5.
Long-term protection of retinal structure and function in RHO-mutant retinas treated with a single vector that combines suppression and replacement of rhodopsin. (A, Upper) Timeline showing time points of injection of scAAV2/5-hOP-RHO820-H1-shRNA820 (5 × 1011 vg/mL titer) in one eye of two RHO-mutant dogs (the contralateral eye was injected with BSS), light exposures (LE1–LE4), OCT imaging, and ERG sessions. (Lower) Representative ONL thickness maps before injection, 11 wk postinjection (immediately before LE1), 1.5 wk post LE1, and 2.1 wk post LE4 of an eye injected with scAAV2/5-hOP-RHO820-H1-shRNA820. Dotted and dashed lines as described in Fig. 4. The optic nerve head (black), major blood vessels (white), tapetum boundary (yellow), and fovea-like region (white ellipse) are overlaid. (Insets) Maps of significance as described in Fig. 3. (B, Left) Schematics showing retinal locations sampled for quantification of ONL thickness and IS/OS intensity within the treated area of two RHO mutant eyes injected with scAAV2/5-hOP-RHO820-H1-shRNA820. (Middle and Right) Longitudinal quantification of the mean (± SD) difference in ONL thickness (Middle) and IS/OS intensity (Right) in the injected eyes compared with uninjected controls. Horizontal dashed lines represent limits of WT variability (± 3 SD). (C) Representative ERG traces of rod [−1.7 log candela (cd)⋅s⋅m−2], mixed rod–cone (0.51 log cd⋅s⋅m−2) recorded in dark-adapted eyes, and cone responses to single stimuli (0.51 log cd⋅s⋅m−2) or to 29-Hz flicker (0.26 log cd⋅s⋅m−2) recorded in light-adapted eyes at ∼2 wk after each of four light-exposure sessions in a RHO-mutant dog injected with scAAV2/5-hOP-RHO820-H1-shRNA820 (green) in one eye and with BSS (red) in the contralateral eye. (D) Longitudinal quantification of maximal amplitudes of mixed rod–cone a- and b-waves (Upper) and of cone responses to 1-Hz and 29-Hz flicker (Lower) in two RHO-mutant dogs injected in one eye with scAAV2/5-RHO820-shRNA820 (green) and in the contralateral eye with BSS (red) at time points similar to those shown in C.
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References

    1. Ginn SL, Alexander IE, Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2012–An update. J Gene Med. 2013;15:65–77. - PubMed
    1. Dunbar CE, et al. Gene therapy comes of age. Science. 2018;359:eaan4672. - PubMed
    1. Ferrua F, Aiuti A. Twenty-five years of gene therapy for ADA-SCID: From bubble babies to an approved drug. Hum Gene Ther. 2017;28:972–981. - PubMed
    1. Kumar SR, Markusic DM, Biswas M, High KA, Herzog RW. Clinical development of gene therapy: Results and lessons from recent successes. Mol Ther Methods Clin Dev. 2016;3:16034. - PMC - PubMed
    1. Mao H, et al. AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa. Hum Gene Ther. 2011;22:567–575. - PMC - PubMed

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