Objectives: With anti-VEGF-based treatments for wet age-related macular degeneration (AMD) requiring frequent injections, it is often burdensome for both patients and healthcare providers. Methods: C57/B6 mice were administered intravitreally with rAAV2-sVEGFRv-1, rAAV2- GFP, or clinically used bevacizumab after CNV lesions were induced via laser photocoagulation. Infiltration by inflammatory cells was significantly decreased by rAAV2-sVEGFRv-1 administration, whereas rAAV2-GFP-treated groups were not.
Overall, rAAV2-sVEGFRv-1 viral vector was favorably comparable to bevacizumab, indicating that it is as effective as approved drugs. Conclusions: The ability of a low dose of rAAV2-sVEGFRv-1 to exert a therapeutically relevant anti-VEGF effect in a CNV model is demonstrated and strongly suggests gene therapy as an effective and convenient treatment for sustained VEGF suppression. Vascular endothelial growth factor (VEGF) has been implicated as a major driver of CNV in wet AMD3, leading to anti-VEGF treatments being widely used as an interventional strategy.
Conventional anti-VEGF treatments include monoclonal antibodies (bevacizumab), Fab fragments (ranibizumab) and recombinant fusion proteins (aflibercept) (table.1)4. The viral vector, rAAV2-sVEGFRv-1, was administered by intravitreal injection (5.0 x 107 v.g.) into the retinas of a mouse model of CNV induced via laser photocoagulation. Subsequent immunostaining of retinal pigment epithelium (RPE)-choroidal mounts revealed that rAAV2-sVEGFRv-1 has anti-angiogenic, anti-inflammatory, anti-fibrotic and anti-apoptotic properties.
When the transgenic construct was expressed, rAAV2-sVEGFRv-1 protected the retina from CNV at levels comparable to bevacizumab treatment.
Materials and methods
The rAAV2-sVEGFRv-1 transgene is controlled by a CMV promoter (▶) and bovine growth hormone poly A signal. Western blotting demonstrated an increase in sVEGFRv-1 protein levels in the culture media after treatment with rAAV2-sVEGFRv-1 (Fig. 5A; n=3), confirming these observations also in rat retinal tissue (data not shown) . The percentage of sVEGFRv secreted indicates that most of the newly synthesized sVEGFRv-1 was secreted into the culture media after treatment with rAAV2-sVEGFRv-1.
In contrast, cells transduced with rAAV2-sVEGFRv-1 were able to resist the angiogenic effects of VEGF, which had fewer cells migrate into the scratch wound among all groups, thus confirming the functionality of the viral vector. As can be seen, when a scratch wound was produced in HUVEC cells expressing rAAV2-sVEGFRv-1 and then treated with VEGF, cell migration into the wound was reduced. This is in contrast to intravitreal injections of rAAV2-sVEGFRv-1, in which CNV formation was markedly suppressed by the injection.
The anti-angiogenic effect of rAAV2-sVEGFRv-1 was comparable to that of bevacizumab, a commonly used drug for the treatment of wet AMD. While CNV was widespread in the sham-treated control mice (A–C) and the group injected with rAAV2-GFP (D–F), CNV was significantly reduced in the groups treated with rAAV2-sVEGFRv-1 (G–I) and bevacizumab (J-L). ). To observe the antifibrotic effects of rAAV2-sVEGFRv-1, transverse retinal sections were immunostained with anti-pan cytokeratin, an indicator of retinal pigment epithelial cell fibrosis.
A marked reduction in apoptotic cells was seen in sections obtained from mice injected with rAAV2-sVEGFRv-1 (G–I) and bevacizumab (J–L), with fewer TUNEL-positive cells being detected in mice subjected to rAAV2-sVEGFRv. - 1 treatment (M); n=3. Here, I show that rAAV2-sVEGFRv-1 is highly capable of exerting anti-VEGF effects to address the core symptoms of wet AMD. Anti-VEGF activity was enhanced in vivo by intravitreal injection of rAAV2-sVEGFRv-1 into the retina of a laser-induced CNV mouse model.
However, rAAV2-sVEGFRv-1 treatment in a mouse model proved that rAAV2-sVEGFRv-1 is also effective in addressing other main symptoms of wet AMD. Meanwhile, immunohistochemistry performed on transverse retinal sections using F4/80 and CD11b, respective markers for macrophages and leukocytes, demonstrated that rAAV2-sVEGFRv-1 had an anti-inflammatory effect. On a symptom-by-symptom basis, rAAV2-sVEGFRv-1 was comparable to bevacizumab in addressing many aspects of wet AMD, and in fact was probably more effective than bevacizumab in reducing leukocyte infiltration in the CNV lesions.
If rAAV2-sVEGFRv-1 were administered to additional model animals, the resulting data sets could show that the effects of the viral vector are virtually indistinguishable from those of bevacizumab. Finally, the strength of rAAV2-sVEGFRv-1 as a potential wet AMD therapeutic becomes apparent when comparing this study with other characterizations of CNV mouse models, particularly those leading to clinical trials. Although rAAV2-sVEGFRv-1 was administered at lower levels (5.0x107v.g.), rAAV2-sVEGFRv-1 had therapeutic effects equivalent to bevacizumab.
In addition, rAAV2-sVEGFRv-1 also outperforms rAAV and sFLT-1-based potential gene therapy for diabetic retinopathy (4.0x1010 v.g.)33.
Adeno-associated viral vector-mediated mTOR inhibition by short hairpin RNA suppresses laser-induced choroidal neovascularization. Potential long-term inhibition of ocular neovascularization by recombinant adeno-associated virus-mediated secretion gene therapy. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1-year follow-up of a phase 1 randomized clinical trial.
Gene therapy in neovascular age-related macular degeneration: three-year follow-up of a phase 1 randomized dose-escalation trial. Constable IJ, Pierce CM, Lai CM, Magno AL, Degli-Esposti MA, French MA, et al. Testa F, Maguire AM, Rossi S, Pierce EA, Melillo P, Marshall K, et al. Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in patients with Leber congenital amaurosis type 2.
Lee SH, Kong YJ, Lyu J, Lee H, Park K, Park TK. Laser photocoagulation induces transduction of retinal pigment epithelial cells by intravitreally administered adeno-associated viral vectors. Igarashi T, Miyake K, Masuda I, Takahashi H, Shimada T. Adeno-associated vector-mediated expression (type 8) of soluble Flt-1 efficiently inhibits neovascularization in a murine choroidal neovascularization model. Ideno J, Mizukami H, Kakehashi A, Saito Y, Okaka T, Urabe M, et al. Prevention of diabetic retinopathy by soluble intraocular flt-1 gene transfer in a spontaneous diabetic mouse model.
AMD 중에서 습성 AMD(wet AMD)는 맥락막 혈관신생(CNV)에 의해 발생하며, 퇴행이 빠르고 치료하지 않으면 영구적인 실명으로 이어질 수 있습니다. 혈관내피성장인자(VEGF)는 습성 AMD에서 CNV의 주요 원인으로 간주되며, 항-VEGF 요법은 중재적 연구로 널리 사용됩니다. 기존의 항-VEGF 치료에는 단일클론 항체(bevacizumab), Fab 단편(ranibizumab), 재조합 융합 단백질(aflibercept)이 사용됩니다.
그러나 이러한 치료법은 항-VEGF 활성을 유지하기 위해 유리체강내 주사를 통해 상대적으로 자주 투여되는데, 이는 절차적 및 경제적 측면 모두에서 부담스러울 수 있습니다. 따라서 항-VEGF 활성을 제공하면서 기존 치료법을 대체할 수 있는 효과적인 방법의 개발이 필요하며, 유전자 치료법이 대안이 될 수 있다. 목적: 습성 AMD에 대한 재조합 아데노 관련 바이러스 2(rAAV2) 전달 시스템에 포장된 가용성 VEGFR-1 변이체(sVEGFRv-1)의 효능과 유전자 치료법 개발 가능성을 검증하는 것입니다.
