Gene delivery to cone photoreceptors by subretinal injection of rAAV2/6 in the mouse retina

Hori, Tesshu; Fukutome, Masashi; Maejima, Chiseto; Matsushima, Hiroki; Kobayashi, Kensuke; Kitazawa, Shigeru; Kitahara, Ryo; Kitano, Katsunori; Kobayashi, Kenta; Moritoh, Satoru; Koike, Chieko

doi:10.1016/j.bbrc.2019.05.117

Cited by 5 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although it is difficult to compare the effective doses from different experiments, this dose between 4 × 10 7 and 4 × 10 8 GC/eye for mice is relatively low, since the effective dose for subretinal injection in mice is typically 1 × 10 8 to 1 × 10 10 GC/eye. 44 , 45 , 46 In this study, AAV8-aflibercept at 4 × 10 8 GC/eye performed worse than AAV8-nVEGFi at 4 × 10 8 GC/eye in reducing CNV areas (63.8% vs. 78.3%, p < 0.05), and it appeared that even AAV8-aflibercept at 4 × 10 9 GC/eye performed worse than AAV8-nVEGFi at 4 × 10 8 GC/eye (67.8% vs. 78.3%), even if the difference was not statistically significant. This is consistent with the expression results in vivo , where the expression of VEGF Trap in AAV8-aflibercept at 4 × 10 9 GC/eye was much lower than that in AAV8-nVEGFi at 4 × 10 8 GC/eye (1,309 ± 695.6 vs. 3,503 ± 856.9 ng/eye).…”

Section: Discussionmentioning

confidence: 99%

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration

She

Wang

et al. 2022

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo . nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro . AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 10 8 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

show abstract

Section: Discussionmentioning

confidence: 99%

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration

She

Wang

et al. 2022

Molecular Therapy - Methods & Clinical Development

View full text Add to dashboard Cite

show abstract

“…Sterile injection was performed according to a previously described method [33][34][35]. In brief, mice were abdominally injected with 1% pentobarbital sodium (50 mg/kg), and tropicamide (0.5%) was dropped on the eyes.…”

Section: Subretinal Injection Of Virusmentioning

confidence: 99%

A de novo missense mutation in MPP2 confers an increased risk of Vogt–Koyanagi–Harada disease as shown by trio-based whole-exome sequencing

Liu,

Meng,

Liao

et al. 2023

Cell Mol Immunol

View full text Add to dashboard Cite

Vogt–Koyanagi–Harada (VKH) disease is a leading cause of blindness in young and middle-aged people. However, the etiology of VKH disease remains unclear. Here, we performed the first trio-based whole-exome sequencing study, which enrolled 25 VKH patients and 50 controls, followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations. A total of 15 de novo mutations in VKH patients were identified, with one of the most important being the membrane palmitoylated protein 2 (MPP2) p.K315N (MPP2-N315) mutation. The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions. Additionally, this mutation appears rare, being absent from the 1000 Genome Project and Genome Aggregation Database, and it is highly conserved in 10 species, including humans and mice. Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis (EAU). In vitro, we used clustered regularly interspaced short palindromic repeats (CRISPR‒Cas9) gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315. Levels of cytokines, such as IL-1β, IL-17E, and vascular endothelial growth factor A, were increased, and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells. Mechanistically, the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315, as shown by LC‒MS/MS and Co-IP, and resulted in activation of the ERK3/IL-17E pathway. Overall, our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.

show abstract

Structural basis for nuclear import of adeno-associated virus serotype 6 capsid protein

Hoad,

Nematollahzadeh,

Petersen

et al. 2024

J Virol

View full text Add to dashboard Cite

Adeno-associated viruses (AAVs) are the most extensively researched viral vectors for gene therapy globally. The AAV viral protein 1 (VP1) N-terminus controls the capsid’s ability to translocate into the cell nucleus; however, the exact mechanism of this process is largely unknown. In this study, we sought to elucidate the precise interactions between AAV serotype 6 (AAV6), a promising vector for immune disorders, and host transport receptors responsible for vector nuclear localization. Focusing on the positively charged basic areas within the N-terminus of AAV6 VP1, we identified a 53-amino acid region that interacts with nuclear import receptors. We measured the binding affinities between this region and various nuclear import receptors, discovering a notably strong interaction with IMPα5 and IMPα7 in the low nanomolar range. We also elucidated the X-ray crystal structure of this region in complex with an importin alpha (IMPα) isoform, uncovering its binding as a bipartite nuclear localization signal (NLS). Furthermore, we show that using this bipartite NLS, AAV6 VP1 capsid protein can localize to the nucleus of mammalian cells in a manner dependent on the IMPα/IMPβ nuclear import pathway. This study provides detailed insights into the interaction between the AAV6 VP1 capsid protein and nuclear import receptors, deepening our knowledge of AAV nuclear import mechanisms and establishing a basis for the improvement of AAV6-based gene therapy vectors. IMPORTANCE AAVs, recognized as the most extensively researched viral vectors for gene therapy globally, offer significant advantages over alternatives due to their small size, non-pathogenic nature, and innate ability for tissue-specific targeting. AAVs are required to localize to the nucleus to perform their role as a gene therapy vector; however, the precise mechanisms that facilitate this process remain unknown. Despite sharing overt genomic similarities with AAV1 and AAV2, AAV6 is a unique serotype. It is currently recognized for its ability to effectively transduce hematopoietic cell lineages and, consequently, is considered promising for the treatment of immune disorders. Identifying the exact mechanisms that permit AAV6 to access the nucleus can open up new avenues for gene therapy vector engineering, which can ultimately lead to increased therapeutic benefits.

show abstract

Gene delivery to cone photoreceptors by subretinal injection of rAAV2/6 in the mouse retina

Cited by 5 publications

References 27 publications

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration

Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration

A de novo missense mutation in MPP2 confers an increased risk of Vogt–Koyanagi–Harada disease as shown by trio-based whole-exome sequencing

Structural basis for nuclear import of adeno-associated virus serotype 6 capsid protein

Contact Info

Product

Resources

About