Safety and distribution evaluation of adeno-associated virus vector 8-<i>PDE6B</i> in mice_Chinese Journal of Ocular Fundus Diseases

Authors：

Qiu Ruiqi ^1,2 , Yang Mingzhu ^1,2 , Jin Xiuxiu ^1,2 , Liu Jingyang ^1,2 , Wang Weiping ^1,2 ,  Lei Bo ^1,2

1. Henan Eye Hospital, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou 450003, China;
2. Eye Institute, Henan Academy of Innovations in Medical Science, Zhengzhou 451100, China;

Corresponding?author：

Lei Bo, Email: bolei99@126.com

Keywords：

Retinitis pigmentosa; PDE6B gene; Adeno-associated virus; Safety evaluation; Vector distribution

DOI：

10.3760/cma.j.cn511434-20251029-00471

Video：

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Objective To observe the ocular and systemic safety and tissue distribution of adeno-associated virus vector 8-PDE6B (AAV8-PDE6B) after subretinal injection in C57BL/6J mice. Methods An animal experimental study. Thirty healthy C57BL/6J mice, 15 females and 15 males, aged 8 weeks, weighing 15-25 g, without specific pathogens, were randomly divided into solvent group, low-dose group, medium-dose group and high-dose group, with 6, 8, 8 and 8 mice respectively. The right eye was used as the experimental eye. Mice in the solvent group were injected with 1 μl of solvent under the retina; mice in the low-dose group, medium-dose group and high-dose group were injected with 1 μl of AAV8-PDE6B vector solution with viral titers of 1.70×10¹², 8.50×10¹² and 4.25×10¹³ vg/ml respectively under the retina. On day 28 after injection, retinal rod cGMP-specific 3',5'-cyclic phosphodiesterase 6β (PDE6B) expression was detected by immunofluorescence staining. Ophthalmic examination, fluorescein angiography (FFA), optical coherence tomography (OCT), hematoxylin-eosin (H&E) staining, and electroretinography (ERG) were performed to evaluate ocular morphology and retinal function. Body weight monitoring, hematological, liver function tests and biochemical analyses, and H&E staining of major tissues were used to evaluate systemic safety. Dot-blot and quantitative real-time polymerase chain reaction (qPCR) were used to detect serum AAV8 neutralizing antibodies and vector distribution, respectively. One-way analysis of variance was used for comparisons among multiple groups. Results Compared with the vehicle group, PDE6B expression in the photoreceptor outer segments was significantly increased in the low-, medium-, and high-dose groups (F=44.027, P＜0.001). On day 28 after injection, no obvious abnormalities were found on ophthalmic examination in any dose group. FFA showed no retinal vascular leakage, and OCT and H&E staining showed intact retinal structure. There were no significant differences in retinal thickness (including total thickness of inner nuclear layer, outer plexiform layer and outer nuclear layer), or ERG amplitudes of dark-adapted a-wave, dark-adapted b-wave, and light-adapted b-wave between the dose groups and the vehicle group (P＞0.05). The mice in all groups remained in good general condition, and no death occurred. There were no significant differences in body weight or major hematological and biochemical parameters among groups (F=1.475, 0.532, 0.285, 1.331, 0.814, 2.199, 2.342, 2.384, 2.650, 2.339, 1.305, 0.555, 1.259; P＞0.05). H&E staining showed no obvious abnormalities in major tissues. No AAV8 neutralizing antibody was detected in serum samples from any group. Except for one spleen sample in the high-dose group with vector gene copy number (11.3 copies/μg DNA) slightly above the lower limit of quantification (10.0 copies/μg DNA), vector gene copy numbers in other major extraocular tissues were below the lower limit of quantification. Conclusion Subretinal injection of low-, medium-, and high-dose AAV8-PDE6B in C57BL/6J mice shows good safety and tolerability, low extraocular tissue distribution, and no obvious humoral immune response.

Citation： Qiu Ruiqi, Yang Mingzhu, Jin Xiuxiu, Liu Jingyang, Wang Weiping, Lei Bo. Safety and distribution evaluation of adeno-associated virus vector 8-PDE6B in mice. Chinese Journal of Ocular Fundus Diseases, 2026, 42(9): 779-788. doi: 10.3760/cma.j.cn511434-20251029-00471 Copy

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2.	Hanany M, Shalom S, Ben-Yosef T, et al. Comparison of worldwide disease prevalence and genetic prevalence of inherited retinal diseases and variant interpretation considerations[J/OL]. Cold Spring Harb Perspect Med, 2024, 14(2): a041277[2024-02-01]. https://pubmed.ncbi.nlm.nih.gov/37460155/. DOI: 10.1101/cshperspect.a041277.
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4.	Kuehlewein L, Zobor D, Stingl K, et al. Clinical phenotype of PDE6B-associated retinitis pigmentosa[J/OL]. Int J Mol Sci, 2021, 22(5): 2374[2021-02-27]. https://pubmed.ncbi.nlm.nih.gov/33673512/. DOI: 10.3390/ijms22052374.
5.	Pearce-Kelling SE, Aleman TS, Nickle A, et al. Calcium channel blocker D-cis-diltiazem does not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis pigmentosa[J/OL]. Mol Vis, 2001, 7: 42-47[2001-02-25]. https://pubmed.ncbi.nlm.nih.gov/11239245/.
6.	Pichard V, Provost N, Mendes-Madeira A, et al. AAV-mediated gene therapy halts retinal degeneration in PDE6beta-deficient dogs[J]. Mol Ther, 2016, 24(5): 867-876. DOI: 10.1038/mt.2016.37.
7.	Han IC, Wiley LA, Ochoa D, et al. Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy[J]. Gene Ther, 2023, 30(3-4): 362-368. DOI: 10.1038/s41434-022-00365-y.
8.	Qiu R, Yang M, Jin X, et al. AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis[J]. Neural Regen Res, 2025, 20(8): 2408-2419. DOI: 10.4103/NRR.NRR-D-23-01301.
9.	He X, Fu Y, Xu Y, et al. A Penetrable AAV2 capsid variant for efficient intravitreal gene delivery to the retina[J/OL]. Invest Ophthalmol Vis Sci, 2025, 66(1): 6[2025-01-02]. https://pubmed.ncbi.nlm.nih.gov/39745676/. DOI: 10.1167/iovs.66.1.6.
10.	Kim HJ, Kwak JH, Choi JS, et al. Subretinal delivery of AAV5-mediated human Pde6b gene ameliorates the disease phenotype in a rat model of retinitis pigmentosa[J]. Mol Vis, 2025, 31: 127-141. DOI: 10.63500/mv_v31_127.
11.	Petit L, Lheriteau E, Weber M, et al. Restoration of vision in the pde6beta-deficient dog, a large animal model of rod-cone dystrophy[J]. Mol Ther, 2012, 20(11): 2019-2030. DOI: 10.1038/mt.2012.134.
12.	Yang M, Qiu R, Wang W, et al. P2X7 receptor antagonist attenuates retinal inflammation and neovascularization induced by oxidized low-density lipoprotein[J/OL]. Oxid Med Cell Longev, 2021, 2021: 5520644[2021-08-19]. https://pubmed.ncbi.nlm.nih.gov/34457115/. DOI: 10.1155/2021/5520644.
13.	Marangoni D, Bush RA, Zeng Y, et al. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice[J/OL]. Mol Ther Methods Clin Dev, 2016, 5: 16011[2016-04-16]. https://pubmed.ncbi.nlm.nih.gov/27626041/. DOI: 10.1038/mtm.2016.11.
14.	She K, Su J, Wang Q, et al. Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration[J]. Mol Ther Methods Clin Dev, 2022, 24: 210-221. DOI: 10.1016/j.omtm.2022.01.002.
15.	Ren D, Fisson S, Dalkara D, et al. Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy[J/OL]. Pharmaceutics, 2022, 14(9): 1973[2022-09-19]. https://pubmed.ncbi.nlm.nih.gov/36145721/. DOI: 10.3390/pharmaceutics14091973.
16.	Irigoyen C, Amenabar Alonso A, Sanchez-Molina J, et al. Subretinal injection techniques for retinal disease: a review[J/OL]. J Clin Med, 2022, 11(16): 4717[2022-08-12]. https://pubmed.ncbi.nlm.nih.gov/36012955/. DOI: 10.3390/jcm11164717.
17.	Ye GJ, Conlon T, Erger K, et al. Safety and biodistribution evaluation of rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus vector expressing retinoschisin, in RS1-deficient mice[J]. Hum Gene Ther Clin Dev, 2015, 26(3): 177-184. DOI: 10.1089/humc.2015.077.
18.	Seitz IP, Michalakis S, Wilhelm B, et al. Superior retinal gene transfer and biodistribution profile of subretinal versus intravitreal delivery of AAV8 in nonhuman primates[J]. Invest Ophthalmol Vis Sci, 2017, 58(13): 5792-5801. DOI: 10.1167/iovs.17-22473.
19.	Kvanta A, Rangaswamy N, Holopigian K, et al. Interim safety and efficacy of gene therapy for RLBP1-associated retinal dystrophy: a phase 1/2 trial[J/OL]. Nat Commun, 2024, 15(1): 7438[2024-09-10]. https://pubmed.ncbi.nlm.nih.gov/39256350/. DOI: 10.1038/s41467-024-51575-4.
20.	McClements ME, Barnard AR, Charbel IP, et al. Assessment of AAV dual vector safety in the Abca4-/- mouse model of Stargardt disease[J/OL]. Transl Vis Sci Technol, 2020, 9(7): 20[2020-06-01]. https://pubmed.ncbi.nlm.nih.gov/32724727/. DOI: 10.1167/tvst.9.7.20.
21.	Qian X, Liu H, Fu S, et al. AAV8-mediated gene therapy rescues retinal degeneration phenotype in a Tlcd3b knockout mouse model[J/OL]. Invest Ophthalmol Vis Sci, 2022, 63(3): 11[2022-03-02]. https://pubmed.ncbi.nlm.nih.gov/35275174/. DOI: 10.1167/iovs.63.3.11.
22.	Swirski S, May O, Ahlers M, et al. In vivo efficacy and safety evaluations of therapeutic splicing correction using U1 snRNA in the mouse retina[J/OL]. Cells, 2023, 12(6): 955[2023-03-21]. https://pubmed.ncbi.nlm.nih.gov/36980294/. DOI: 10.3390/cells12060955.
23.	He X, Fu Y, Ma L, et al. AAV for gene therapy in ocular diseases: progress and prospects[J/OL]. Research (Wash D C), 2023, 6: 0291[2023-12-22]. https://pubmed.ncbi.nlm.nih.gov/38188726/. DOI: 10.34133/research.0291.
24.	Han IC, Cheng JL, Burnight ER, et al. Retinal tropism and transduction of adeno-associated virus varies by serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats[J]. Hum Gene Ther, 2020, 31(23-24): 1288-1299. DOI: 10.1089/hum.2020.043.
25.	Yiu G, Chung SH, Mollhoff IN, et al. Suprachoroidal and subretinal injections of AAV using transscleral microneedles for retinal gene delivery in nonhuman primates[J]. Mol Ther Methods Clin Dev, 2020, 16: 179-191. DOI: 10.1016/j.omtm.2020.01.002.
26.	Wiley LA, Boyce TM, Meyering EE, et al. The degree of adeno-associated virus-induced retinal inflammation varies based on serotype and route of delivery: intravitreal, subretinal, or suprachoroidal[J]. Hum Gene Ther, 2023, 34(11-12): 530-539. DOI: 10.1089/hum.2022.222.
27.	Song L, Ren C, Luo M, et al. Retinal pigment epithelium-targeting gene therapy corrects ocular symptoms in mouse and rat models of oculocutaneous albinism type I[J/OL]. MedComm (2020), 2025, 6(11): e70433[2025-10-20]. https://pubmed.ncbi.nlm.nih.gov/41122448/. DOI: 10.1002/mco2.70433.

1. Liu Y, Zong X, Cao W, et al. Gene therapy for retinitis pigmentosa: current challenges and new progress[J/OL]. Biomolecules, 2024, 14(8): 903[2024-07-25]. https://pubmed.ncbi.nlm.nih.gov/39199291/. DOI: 10.3390/biom14080903.
2. Hanany M, Shalom S, Ben-Yosef T, et al. Comparison of worldwide disease prevalence and genetic prevalence of inherited retinal diseases and variant interpretation considerations[J/OL]. Cold Spring Harb Perspect Med, 2024, 14(2): a041277[2024-02-01]. https://pubmed.ncbi.nlm.nih.gov/37460155/. DOI: 10.1101/cshperspect.a041277.
3. Kim MS, Joo K, Seong MW, et al. Genetic mutation profiles in Korean patients with inherited retinal diseases[J/OL]. J Korean Med Sci, 2019, 34(21): e161[2019-06-02]. https://pubmed.ncbi.nlm.nih.gov/31144483/. DOI: 10.3346/jkms.2019.34.e161.
4. Kuehlewein L, Zobor D, Stingl K, et al. Clinical phenotype of PDE6B-associated retinitis pigmentosa[J/OL]. Int J Mol Sci, 2021, 22(5): 2374[2021-02-27]. https://pubmed.ncbi.nlm.nih.gov/33673512/. DOI: 10.3390/ijms22052374.
5. Pearce-Kelling SE, Aleman TS, Nickle A, et al. Calcium channel blocker D-cis-diltiazem does not slow retinal degeneration in the PDE6B mutant rcd1 canine model of retinitis pigmentosa[J/OL]. Mol Vis, 2001, 7: 42-47[2001-02-25]. https://pubmed.ncbi.nlm.nih.gov/11239245/.
6. Pichard V, Provost N, Mendes-Madeira A, et al. AAV-mediated gene therapy halts retinal degeneration in PDE6beta-deficient dogs[J]. Mol Ther, 2016, 24(5): 867-876. DOI: 10.1038/mt.2016.37.
7. Han IC, Wiley LA, Ochoa D, et al. Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy[J]. Gene Ther, 2023, 30(3-4): 362-368. DOI: 10.1038/s41434-022-00365-y.
8. Qiu R, Yang M, Jin X, et al. AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis[J]. Neural Regen Res, 2025, 20(8): 2408-2419. DOI: 10.4103/NRR.NRR-D-23-01301.
9. He X, Fu Y, Xu Y, et al. A Penetrable AAV2 capsid variant for efficient intravitreal gene delivery to the retina[J/OL]. Invest Ophthalmol Vis Sci, 2025, 66(1): 6[2025-01-02]. https://pubmed.ncbi.nlm.nih.gov/39745676/. DOI: 10.1167/iovs.66.1.6.
10. Kim HJ, Kwak JH, Choi JS, et al. Subretinal delivery of AAV5-mediated human Pde6b gene ameliorates the disease phenotype in a rat model of retinitis pigmentosa[J]. Mol Vis, 2025, 31: 127-141. DOI: 10.63500/mv_v31_127.
11. Petit L, Lheriteau E, Weber M, et al. Restoration of vision in the pde6beta-deficient dog, a large animal model of rod-cone dystrophy[J]. Mol Ther, 2012, 20(11): 2019-2030. DOI: 10.1038/mt.2012.134.
12. Yang M, Qiu R, Wang W, et al. P2X7 receptor antagonist attenuates retinal inflammation and neovascularization induced by oxidized low-density lipoprotein[J/OL]. Oxid Med Cell Longev, 2021, 2021: 5520644[2021-08-19]. https://pubmed.ncbi.nlm.nih.gov/34457115/. DOI: 10.1155/2021/5520644.
13. Marangoni D, Bush RA, Zeng Y, et al. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice[J/OL]. Mol Ther Methods Clin Dev, 2016, 5: 16011[2016-04-16]. https://pubmed.ncbi.nlm.nih.gov/27626041/. DOI: 10.1038/mtm.2016.11.
14. She K, Su J, Wang Q, et al. Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration[J]. Mol Ther Methods Clin Dev, 2022, 24: 210-221. DOI: 10.1016/j.omtm.2022.01.002.
15. Ren D, Fisson S, Dalkara D, et al. Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy[J/OL]. Pharmaceutics, 2022, 14(9): 1973[2022-09-19]. https://pubmed.ncbi.nlm.nih.gov/36145721/. DOI: 10.3390/pharmaceutics14091973.
16. Irigoyen C, Amenabar Alonso A, Sanchez-Molina J, et al. Subretinal injection techniques for retinal disease: a review[J/OL]. J Clin Med, 2022, 11(16): 4717[2022-08-12]. https://pubmed.ncbi.nlm.nih.gov/36012955/. DOI: 10.3390/jcm11164717.
17. Ye GJ, Conlon T, Erger K, et al. Safety and biodistribution evaluation of rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus vector expressing retinoschisin, in RS1-deficient mice[J]. Hum Gene Ther Clin Dev, 2015, 26(3): 177-184. DOI: 10.1089/humc.2015.077.
18. Seitz IP, Michalakis S, Wilhelm B, et al. Superior retinal gene transfer and biodistribution profile of subretinal versus intravitreal delivery of AAV8 in nonhuman primates[J]. Invest Ophthalmol Vis Sci, 2017, 58(13): 5792-5801. DOI: 10.1167/iovs.17-22473.
19. Kvanta A, Rangaswamy N, Holopigian K, et al. Interim safety and efficacy of gene therapy for RLBP1-associated retinal dystrophy: a phase 1/2 trial[J/OL]. Nat Commun, 2024, 15(1): 7438[2024-09-10]. https://pubmed.ncbi.nlm.nih.gov/39256350/. DOI: 10.1038/s41467-024-51575-4.
20. McClements ME, Barnard AR, Charbel IP, et al. Assessment of AAV dual vector safety in the Abca4-/- mouse model of Stargardt disease[J/OL]. Transl Vis Sci Technol, 2020, 9(7): 20[2020-06-01]. https://pubmed.ncbi.nlm.nih.gov/32724727/. DOI: 10.1167/tvst.9.7.20.
21. Qian X, Liu H, Fu S, et al. AAV8-mediated gene therapy rescues retinal degeneration phenotype in a Tlcd3b knockout mouse model[J/OL]. Invest Ophthalmol Vis Sci, 2022, 63(3): 11[2022-03-02]. https://pubmed.ncbi.nlm.nih.gov/35275174/. DOI: 10.1167/iovs.63.3.11.
22. Swirski S, May O, Ahlers M, et al. In vivo efficacy and safety evaluations of therapeutic splicing correction using U1 snRNA in the mouse retina[J/OL]. Cells, 2023, 12(6): 955[2023-03-21]. https://pubmed.ncbi.nlm.nih.gov/36980294/. DOI: 10.3390/cells12060955.
23. He X, Fu Y, Ma L, et al. AAV for gene therapy in ocular diseases: progress and prospects[J/OL]. Research (Wash D C), 2023, 6: 0291[2023-12-22]. https://pubmed.ncbi.nlm.nih.gov/38188726/. DOI: 10.34133/research.0291.
24. Han IC, Cheng JL, Burnight ER, et al. Retinal tropism and transduction of adeno-associated virus varies by serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats[J]. Hum Gene Ther, 2020, 31(23-24): 1288-1299. DOI: 10.1089/hum.2020.043.
25. Yiu G, Chung SH, Mollhoff IN, et al. Suprachoroidal and subretinal injections of AAV using transscleral microneedles for retinal gene delivery in nonhuman primates[J]. Mol Ther Methods Clin Dev, 2020, 16: 179-191. DOI: 10.1016/j.omtm.2020.01.002.
26. Wiley LA, Boyce TM, Meyering EE, et al. The degree of adeno-associated virus-induced retinal inflammation varies based on serotype and route of delivery: intravitreal, subretinal, or suprachoroidal[J]. Hum Gene Ther, 2023, 34(11-12): 530-539. DOI: 10.1089/hum.2022.222.
27. Song L, Ren C, Luo M, et al. Retinal pigment epithelium-targeting gene therapy corrects ocular symptoms in mouse and rat models of oculocutaneous albinism type I[J/OL]. MedComm (2020), 2025, 6(11): e70433[2025-10-20]. https://pubmed.ncbi.nlm.nih.gov/41122448/. DOI: 10.1002/mco2.70433.

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Chinese Journal of Ocular Fundus Diseases

Safety and distribution evaluation of adeno-associated virus vector 8-PDE6B in mice

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