AAV-PHP.eB transduces both the inner and outer retina with high efficacy in mice
2022; Cell Press; Volume: 25; Linguagem: Inglês
10.1016/j.omtm.2022.03.016
ISSN2329-0501
AutoresArpad Palfi, Naomi Chadderton, Sophia Millington‐Ward, Iris J. M. Post, Pete Humphries, Paul F. Kenna, G. Jane Farrar,
Tópico(s)CRISPR and Genetic Engineering
ResumoRecombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches; however, there is a need to further improve the efficacy, tropism, and safety of these vectors. In this study, using a CMV-EGFP expression cassette, we characterize the retinal utility of AAV-PHP.eB, a serotype recently developed by in vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in the high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50–70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate the pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina. Recombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches; however, there is a need to further improve the efficacy, tropism, and safety of these vectors. In this study, using a CMV-EGFP expression cassette, we characterize the retinal utility of AAV-PHP.eB, a serotype recently developed by in vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in the high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50–70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate the pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina. IntroductionWith more than 30 completed or ongoing clinical trials (ClinicalTrials.gov) and many more in the pipeline, AAV-delivered retinal gene therapies promise treatments for visual impairments that are untreatable with existing conventional therapeutics. Data regarding safety and efficacy of AAV-gene therapies have accrued rapidly and can be exemplified with the success of Luxturna, the first authorized retinal gene therapy targeting RPE (retinal pigment epithelium)65-linked retinal dystrophy.1Maguire A.M. Russell S. Wellman J.A. Chung D.C. Yu Z.-F. Tillman A. Wittes J. Pappas J. Elci O. Marshall K.A. et al.Efficacy, safety, and durability of voretigene neparvovecrzyl in RPE65 mutation-associated inherited retinal dystrophy: results of phase 1 and 3 trials.Ophthalmology. 2019; 126: 1273-1285Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,2Russell S. Bennett J. Wellman J.A. Chung D.C. Yu Z.-F. Tillman A. Wittes J. Pappas J. Elci O. 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For example, AAV2/2 is highly efficient at transducing retinal ganglion cells (RGCs) via intravitreal (IVT) injection.18Chadderton N. Palfi A. Millington-Ward S. Gobbo O. Overlack N. Carrigan M. O'Reilly M. Campbell M. Ehrhardt C. Wolfrum U. et al.Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.Eur. J. Hum. Genet. 2013; 21: 62-68Crossref PubMed Scopus (55) Google Scholar, 19Chaffiol A. Caplette R. Jaillard C. Brazhnikova E. Desrosiers M. Dubus E. Duhamel L. Macé E. Marre O. Benoit P. et al.A new promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retina.Mol. Ther. 2017; 25: 2546-2560Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 20Vandenberghe L.H. Bell P. Maguire A.M. Cearley C.N. Xiao R. Calcedo R. Wang L. Castle M.J. Maguire A.C. Grant R. et al.Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey.Sci. 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Biol. 2019; 1185: 109-112Crossref PubMed Scopus (5) Google Scholar A limited number of RGC (IVT delivery) and some cells in the bipolar and ganglion cell layers (systemic delivery) were also transduced, suggesting that AAV-PHP.eB can target various retinal cell types, albeit depending on the route of administration.35Simpson C.P. Bolch S.N. Zhu P. Weidert F. Dinculescu A. Lobanova E.S. Systemic delivery of genes to retina using adeno-associated viruses.Adv. Exp. Med. Biol. 2019; 1185: 109-112Crossref PubMed Scopus (5) Google Scholar As the original work with AAV-PHP.eB in the mouse retina was a proof-of-concept study with limited data presented, we decided to further explore the utility of this serotype in retinal gene delivery.35Simpson C.P. Bolch S.N. Zhu P. Weidert F. Dinculescu A. Lobanova E.S. Systemic delivery of genes to retina using adeno-associated viruses.Adv. Exp. Med. Biol. 2019; 1185: 109-112Crossref PubMed Scopus (5) Google Scholar Utilizing the AAV-PHP.eB capsid, we delivered a cytomegalovirus (CMV) promoter-driven EGFP expression cassette (CMV-EGFP) via systemic, IVT, and SR routes to the murine retina. We compared the tropism and efficacy of AAV-PHP.eB to IVT and SR deliveries of AAV2/236Hordeaux J. Yuan Y. Clark P.M. Wang Q. Martino R.A. Sims J.J. Bell P. Raymond A. Stanford W.L. Wilson J.M. The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier.Mol. Ther. 2019; 27: 912-921Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 37Dudus L. Anand V. Acland G.M. Chen S.J. Wilson J.M. Fisher K.J. Maguire A.M. Bennett J. Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV.Vis. Res. 1999; 39: 2545-2553Crossref PubMed Scopus (138) Google Scholar, 38Harvey A.R. Kamphuis W. Eggers R. Symons N.A. Blits B. Niclou S. Boer G.J. Verhaagen J. 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Ther. 2011; 19: 260-265Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar capsids, respectively, which are highly efficient at transducing the retina via these routes.ResultsIn this study, we assessed the utility of the recently derived AAV-PHP.eB serotype for gene delivery to the retina.23Chan K.Y. Jang M.J. Yoo B.B. Greenbaum A. Ravi N. Wu W.-L. Sánchez-Guardado L. Lois C. Mazmanian S.K. Deverman B.E. et al.Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.Nat. Neurosci. 2017; 20: 1172-1179Crossref PubMed Scopus (500) Google Scholar We analyzed EGFP expression in the murine retina transduced with AAV-PHP.eB-CMV-EGFP using different delivery routes, including systemic delivery via tail vein (TV) injection, as well as IVT and SR intraocular injections. Control AAVs with serotypes frequently used for intraocular administration, that is, AAV2/2-CMV-EGFP for IVT injection, and AAV2/8-CMV-EGFP for SR injection were used to enable the comparative analysis of the different serotypes.Systemic and intravitreal deliveryAdult 129 S2/SvHsd mice were used in this study as the 129 strains were permissive to PHP.B transduction across the blood-brain barrier.36Hordeaux J. Yuan Y. Clark P.M. Wang Q. Martino R.A. Sims J.J. Bell P. Raymond A. Stanford W.L. Wilson J.M. The GPI-linked protein LY6A drives AAV-PHP.B transport across the blood-brain barrier.Mol. Ther. 2019; 27: 912-921Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Mice were administered with 5.0 × 1010 vg of AAV-PHP.eB-CMV-EGFP via TV injection (TV AAV-PHP.eB-CMV-EGFP) or 7.5 × 108 vg/eye of AAV-PHP.eB-CMV-EGFP or AAV2/2-CMV-EGFP via IVT injection (IVT AAV-PHP.eB-CMV-EGFP and IVT AAV2/2-CMV-EGFP, respectively; n = 3–4). AAV2/2 was selected as a control serotype as it is the serotype most extensively used for IVT delivery to RGCs in rodents, NHPs, and humans.18Chadderton N. Palfi A. Millington-Ward S. Gobbo O. Overlack N. Carrigan M. O'Reilly M. Campbell M. Ehrhardt C. Wolfrum U. et al.Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.Eur. J. Hum. Genet. 2013; 21: 62-68Crossref PubMed Scopus (55) Google Scholar,37Dudus L. Anand V. Acland G.M. Chen S.J. Wilson J.M. Fisher K.J. Maguire A.M. Bennett J. Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV.Vis. Res. 1999; 39: 2545-2553Crossref PubMed Scopus (138) Google Scholar, 38Harvey A.R. Kamphuis W. Eggers R. Symons N.A. Blits B. Niclou S. Boer G.J. Verhaagen J. Intravitreal injection of adeno-associated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: a comparison with lentiviral vectors.Mol. Cell Neurosci. 2002; 21: 141-157Crossref PubMed Scopus (103) Google Scholar, 39Lukason M. DuFresne E. Rubin H. Pechan P. Li Q. Kim I. Kiss S. Flaxel C. Collins M. Miller J. et al.Inhibition of choroidal neovascularization in a nonhuman primate model by intravitreal administration of an AAV2 vector expressing a novel anti-VEGF molecule.Mol. Ther. 2011; 19: 260-265Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 40Feuer W.J. Schiffman J.C. Davis J.L. Porciatti V. Gonzalez P. Koilkonda R.D. Yuan H. Lalwani A. Lam B.L. Guy J. Gene therapy for leber hereditary optic neuropathy: initial results.Ophthalmology. 2016; 123: 558-570Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 41Newman N.J. Yu-Wai-Man P. Carelli V. Moster M.L. Biousse V. Vignal-Clermont C. Sergott R.C. Klopstock T. Sadun A.A. Barboni P. et al.Efficacy and safety of intravitreal gene therapy for leber hereditary optic neuropathy treated within 6 Months of disease onset.Ophthalmology. 2021; 128: 649-660Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar As AAV2/2 does not cross the blood-brain barrier, it was not administered via TV.42Zhang H. Yang B. Mu X. Ahmed S.S. Su Q. He R. Wang H. Mueller C. Sena-Esteves M. Brown R. et al.Several rAAV vectors efficiently cross the blood–brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system.Mol. Ther. 2011; 19: 1440-1448Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar The effective dose of AAV-PHP.eB-CMV-EGFP was based on previous studies,23Chan K.Y. Jang M.J. Yoo B.B. Greenbaum A. Ravi N. Wu W.-L. Sánchez-Guardado L. Lois C. Mazmanian S.K. Deverman B.E. et al.Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.Nat. Neurosci. 2017; 20: 1172-1179Crossref PubMed Scopus (500) Google Scholar,35Simpson C.P. Bolch S.N. Zhu P. Weidert F. Dinculescu A. Lobanova E.S. Systemic delivery of genes to retina using adeno-associated viruses.Adv. Exp. Med. Biol. 2019; 1185: 109-112Crossref PubMed Scopus (5) Google Scholar,43Chai Z. Sun J. Rigsbee K.M. Wang M. Samulski R.J. Li C. Application of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody evasion.J. Control Release. 2017; 262: 348-356Crossref PubMed Scopus (18) Google Scholar,44Zincarelli C. Soltys S. Rengo G. Rabinowitz J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.Mol. Ther. 2008; 16: 1073-1080Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar and as it reflected dosage to the whole body, it was significantly higher than the dose range used for direct IVT delivery to the eye.18Chadderton N. Palfi A. Millington-Ward S. Gobbo O. Overlack N. Carrigan M. O'Reilly M. Campbell M. Ehrhardt C. Wolfrum U. et al.Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.Eur. J. Hum. Genet. 2013; 21: 62-68Crossref PubMed Scopus (55) Google Scholar EGFP expression from transduced retinas was analyzed by histology at 1 month post-AAV delivery (Figure 1). With both AAVs and delivery routes, significant EGFP expression was found in the inner retina (Figure 1), while very few labeled cells were detected in the outer retina (for the latter, an example is given in Figure 1D). If present, outer retina labeling was mostly detected in areas with very high transduction rates with IVT delivery. Notably, TV AAV-PHP.eB-CMV-EGFP resulted in an even transduction of the whole murine retina (Figure 1A). In contrast, IVT AAV-PHP.eB-CMV-EGFP and IVT AAV2/2-CMV-EGFP resulted in partial and uneven transduction of the retina (Figures 1B and 1C). The observed significant transduction via IVT AAV-PHP.eB-CMV-EGFP (Figure 1B) was unexpected as minimal transduction was observed via IVT AAV-PHP.eB previously.35Simpson C.P. Bolch S.N. Zhu P. Weidert F. Dinculescu A. Lobanova E.S. Systemic delivery of genes to retina using adeno-associated viruses.Adv. Exp. Med. Biol. 2019; 1185: 109-112Crossref PubMed Scopus (5) Google Scholar EGFP fluorescence in cells in the ganglion cell layer (GCL) was typically less intense in TV AAV-PHP.eB-CMV-EGFP transduced retinas (Figures 1D–1F). In the transduced areas, the number of EGFP+ cells in the GCL were slightly higher in the eyes transduced via IVT (IVT AAV-PHP.eB-CMV-EGFP: 60.6 ± 6.2 cells/mm, p < 0.05, ANOVA; IVT AAV2/2-CMV-EGFP: 53.4 ± 5.4 cells/mm, p = 0.19, ANOVA; Figure 1G) versus TV (TV AAV-PHP.eB-CMV-EGFP: 42.1 ± 10.6 cells/mm; Figure 1G) delivery. Similarly, the number of EGFP positive cells in the inner nuclear layer (INL) were also higher in the eyes transduced via IVT (IVT AAV-PHP.eB-CMV-EGFP: 79.6 ± 15.1 cells/mm, p = 0.19, ANOVA; IVT AAV2/2-CMV-EGFP: 89.7 ± 11.8 cells/mm, p < 0.05, ANOVA; Figure 1H) versus TV (TV AAV-PHP.eB-CMV-EGFP: 62.0 ± 9.1 cells/mm; Figure 1H) delivery. The difference in both EGFP intensities in cells and the EGFP+ cell numbers between TV and IVT injections may, in part, be a function of the doses delivered. It is therefore reasonable to assume that a higher TV dose could have resulted in higher EGFP intensities and number of EGFP+ cells. Meanwhile, the similar number of transduced cells between IVT AAV-PHP.eB-CMV-EGFP and IVT AAV2/2-CMV-EGFP deliveries (using the same dose) suggest similar efficacies for these serotypes. The ratios of the EGFP+ cells in the INL versus GCL were also similar for the three delivery combinations (Figure 1I), suggesting a generally similar tropisms for these AAVs and delivery routes.To identify the retinal cell type of the EGFP+ cells, immunocytochemistry with various retinal cell markers, including RBPMS (RGC marker), PAX6 (RGC and amacrine cell marker), VSX2 (bipolar cell marker), and CRALBP (Müller cell marker) was carried out on the transduced retinal sections (Figure 2 and Table 1). For both routes of administration, TV and IVT, the RGCs (RBPMS+ cells) were efficiently transduced with AAV-PHP.eB-CMV-EGFP at the used doses (Figures 2A–2F); most of the labeled cells in the GCL were RGCs. RGC axons and dendrites were also labeled following both TV and IVT delivery (Figures 2A–2F; see also Figures 1D–1F). Colabelling revealed that some amacrine cells (PAX6+) and bipolar cells (VSX2+) were transduced in the INL in a similar fashion by TV AAV-PHP.eB-CMV-EGFP, IVT AAV-PHP.eB-CMV-EGFP, and IVT AAV2/2-CMV-EGFP (Figures 2G–2L). Horizontal cells (identified by their localization, shape, and arborization) were also targeted; the highest transduction level was found in retinas receiving TV AAV-PHP.eB-CMV-EGFP (Figures 2G–2I and 2M–2O). Müller cells (CRALBP+) were not transduced at all using either route of administration (Figures 2M–2R).Figure 2Colabeling of EGFP transduced cells with cell markers in the inner retinaShow full captionAdult mice received 5.0 × 1010 vg/animal of AAV-PHP.eB-CMV-EGFP via TV injection or 7.5 × 108 vg/eye of AAV-PHP.eB-CMV-EGFP or AAV2/2-CMV-EGFP (control) IVT (n = 3–4). Eyes were enucleated, fixed in 4% PFA, cryosectioned, and stained with immunohistochemistry for retinal cell markers at 1 month post-delivery. (A–F) GCL. (A–C) EGFP fluorescence (green), (D–F) overlay of EGFP fluorescence (green), RBPMS immunohistochemistry (retinal ganglion cell marker, magenta), and nuclear counterstain (DAPI, blue). Arrowheads indicate examples of EGFP+ retinal ganglion cells. (G–R) INL. (G–I) EGFP fluorescence (green), (J–L) overlay of EGFP fluorescence (green), VSX2 immunohistochemistry (bipolar cell marker; magenta), and PAX6 immunohistochemistry (amacrine cell marker; light blue). Upward arrowheads indicate examples of EGFP+ bipolar cells; downward arrowheads indicate examples of EGFP+ amacrine cells, while arrows indicate examples of EGFP+ horizontal cells. (M–O) EGFP fluorescence (green). (P–R) Overlay of EGFP fluorescence (green) and CRALBP immunohisto
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