These results reflected quantifications of retinal avascular area and blood vessel tortuosity, with rAAV2-shmTOR-GFP showing significantly greater therapeutic efficacy than the other treatments. In addition, the virus vector was shown to reduce macrophage infiltration into retinal tissue and possess anti-apoptotic properties, with both processes associated with wet AMD. Taken together, these results build on the previous work to further demonstrate the strong promise of rAAV2-shmTOR-GFP as an effective and convenient gene therapy for wet AMD.
However, since this downregulation of VEGF is partial,14 the development of gene therapeutics for wet AMD targeting mTOR requires inhibition of all three complexes. All of these aspects are associated with wet AMD and further demonstrate the promise of rAAV2-shmTOR-GFP as a potential gene therapeutic to treat the condition.
Materials and Methods
However, due to the lower levels of neovascularization observed in rats compared to the mouse OIR model,20 in particular Sprague Dawley rats,22 the pups were placed under hyperoxia on postnatal day 4 (P4) to take advantage of the less developed retinal vasculature and induce a hyperoxia. more dramatic vaso-destruction, as neovascularization depends on the extent of vaso-destruction.23 At P9, pups were returned to normoxia and either injected intravitreally with their respective virus vectors, subjected to a sham treatment, or left untreated before euthanization at P14 . The experimental virus vectors, rAAV2-shmTOR-GFP (formerly rAAV-mTOR shRNA-EGFP) and rAAV2-shCon-GFP (formerly rAAV-encoded shRNA-EGFP), were prepared as previously described.19 All virus vectors used in this research paper was obtained from CdmoGen Co., Ltd. PFA in 0.1 M PB and the anterior sections were then removed, including the cornea and lens, to generate eyecups.
Neovascularization was determined by manually outlining the areas where neovascular bundles formed and comparing the sum of their sum to the total area of the flat mount. Vessel rotation was determined by drawing a straight line from the end of a retinal artery to where it begins toward the center of the retina. San Francisco, CA) or anti-mTOR (AF15371; R&D Systems; Minneapolis, MN) by incubating the samples overnight at 4°C with the diluted primary antibodies.
They were then washed 3 times in PBST for 10 min each, incubated for 2 h at room temperature with Alexa Fluor 568 or 488 secondary antibodies (Thermo Fisher Scientific), and stained with DAPI (D9542; Sigma-Aldrich) to visualize the cell. nuclei. The TUNEL assay was performed according to the Roche Diagnostics protocol; . Indianapolis, IN), retinal sections were then washed 3 times in PBST for 10 min per piece and counterstained with DAPI. The samples were then examined with an LSM 700 fluorescence confocal microscope (Carl Zeiss Microscopy; Jena, Germany) and images were captured using ImageJ.
The oxygen-induced retinopathy model exploits the underdeveloped retinal vascular system of rats at birth.
Results
After 48 h, total RNA was isolated for RT-qPCR analysis (B) or whole-cell lysates prepared for immunoblotting (C), showing that rAAV2-shmTOR-GFP treatment significantly reduced mTOR mRNA and protein expression levels, respectively, in vitro. Immunohistochemistry performed on frozen sections using an antibody for GFP revealed that rAAVs delivered via intravitreal injections are able to efficiently transduce rat retinas since GFP was expressed in animals treated with the experimental viral vectors (Fig. 2L, 2P) and absent in the normal control (Fig. 2D) and mock-treated (Fig. 2H) groups. Immunostaining with anti-mTOR showed that the rat OIR model was successfully generated, with mock-treated (Fig. 2I-2K) and rAAV2-shCon-GFP-injected (Fig. 2M-2O) control groups exhibiting elevated mTOR -protein levels compared to rats not exposed to hyperoxia (Fig. 2E-2G).
In addition, the almost complete downregulation of mTOR expression (Fig. 2Q-2S) confirmed the in vivo activity of rAAV2-shmTOR-GFP. The implications of this mTOR inhibitory activity, as it relates to AMD, were investigated by means of fluorescence angiography ( Fig. 3A–3E ). In rat OIR models, the return of the pups to normoxia after vaso-extinction occurred during the hyperoxic phase should lead to the sprouting of new capillaries.
During vaso-obliteration in the first part of OIR model generation, the creation of a capillary-free area in the rat retina results in an increased amount of avascular areas,25 and as vessels regenerate in the second phase, an inverse relationship is found. to exist between neovascular tufting and healthy vascular regeneration.24 As such, a reduction in neovascularization correlates to increased normal revascularization, resulting in decreased avascularity. While vascular sprouting from the latter and any unaffected capillaries are responsible for regenerating the capillary network, with neovascular tufting being a dysfunction of this process, retinal arteries become tortuous, which is found in a variety of retinopathic conditions. 24 Map tortuosity was least severe in rats treated with rAAV2- shmTOR-GFP p=0.00040; n=5), whereas ratios between and were observed in untreated, mock-treated and rAAV2-shCon-GFP-treated animals (n=5) (Fig. 3H), respectively. Immunohistochemistry using anti-F4/80, which indicates the presence of macrophages, revealed significant inflammatory cell infiltration in the retinas of untreated (D-F), mock-treated (G-I) and rAAV2-shCon-GFP-treated (J-L) OIR model groups .
Due to the known association of inflammatory processes with the initiation and progression of wet AMD, the infiltration of macrophages into the transverse retinal sections was determined by anti-F4/80 immunostaining (Fig. 4). TUNEL-positive cells could not be found in the normal control group (A-C), which was not exposed to hyperoxic conditions, but were detected in transverse retinal sections taken from rats that were untreated (D-F), sham-treated (G-I), or injected with rAAV2-shCon-GFP (JL). Significantly fewer apoptotic cells were found in the OIR model group treated with rAAV2-shmTOR-GFP (M-O), which was quantified (P; n = 5), indicating that mTOR inhibition through RNA interference has anti-apoptotic activity.
Discussion
Furthermore, RPE cells can undergo oxidative stress-induced senescence.29 In the aging retina, reactive oxygen species (ROS) act as a trigger for parainflammation.30 This chronic condition may be directly linked to AMD initiation and/or progression,30 as persistent inflammation. contributes to several age-related diseases, whereas sporadic inflammation serves as a crucial survival mechanism.28 As an age-related condition, cellular senescence is one of the major causal links to AMD, although this has not yet been thoroughly investigated.2 In all studies with non-human animal species to date, mTOR inhibition has been shown to extend lifespan while delaying age-related diseases in mice.31. In old cells affected by degeneration, especially post-mitotic RPE cells, autophagy capacity is reduced27 and this reduction has been linked to AMD pathogenesis.29 As a critical component of the autophagy pathway, 10 mTOR inhibition may be therapeutically beneficial. Like autophagy dysfunction, inflammation is implicated in all stages of AMD,2 as inflammation-related proteins can be found in drusen,28 one of the defining characteristics of early- and intermediate-stage AMD.2Inflammation was also shown in mouse OIR models to play a role in neovascular tufting, as the extent of it occurred was reduced via anti-.
Cytokines including IL-1β and TNF-α were also implicated in endothelial cell activation during early stages of inflammation, indicating that mTOR inhibition may also involve an anti-angiogenic component.19 Although neovascularization may be the defining feature of wet AMD, the anti-VEGF therapies developed so far seem limited to treating just the most obvious presentation of the condition and not wet AMD as a whole. In addition to those described above, immunological30 and fibrotic34 processes are also known to be involved in wet AMD. Until recently, studies involving mTOR inhibition for the treatment of wet AMD have generally used rapalogs,35 which affect only mTORC1.
This results in the increased expression of VEGF,14 one of the drivers of neovascular tufting.24 VEGF is also a target gene of HIF-2α, the expression of which is enhanced via the stabilization and activation of mTORC2,36 meaning it is unaffected by rapamycin or its analogues. However, activation of the HIF-2α pathway can also upregulate the expression of SLC7A5, an activator of mTORC1, directly increasing mTORC1, HIF-1α, and VEGF activity.37 Treatment with pp242, an mTOR inhibitor of the second generation, significantly reduces both HIF-2α mRNA levels and VEGF expression,36 demonstrating that mTORC1 inhibition is only limited in anti-angiogenic capabilities. Another mechanism by which mTOR evades the inhibition of mTORC1 is through Akt, the main phosphorylation target of mTORC2.10 Rapamycin and its analogs temsirolimus38 and everolimus39 successfully inhibited mTORC1 both in vivo and in vitro, but this also led to the activation of Akt, a component of the insulin/PI3K pathway responsible for mTORC1 activation.40 While second-generation mTOR inhibitors have demonstrated their effectiveness in downregulating mTORC1, mTORC2, and even mTORC3 activity,9 long-term treatment is not capable of eventual reactivation of Akt via the insulin/PI3K pathway.10.
As such, direct targeting via RNA interference is the most reliable method to ensure the comprehensive inhibition of mTOR, a promising therapeutic target for wet AMD.
Temsirolimus inhibits proliferation and migration in retinal pigment epithelial and endothelial cells via mTOR inhibition and reduces VEGF and PDGF expression. Rapamycin reduces VEGF expression in retinal pigment epithelium (RPE) and inhibits RPE-induced sprout angiogenesis in vitro. Multispecies compatible antitumor effects of a cross-species small interfering RNA against mammalian target of rapamycin.
Prospects for retinal gene therapy after commercialization of the voretigene neparvovec-rzyl for retinal dystrophy mediated by RPE65 mutation. Adeno-associated viral vector-mediated mTOR inhibition by short hairpin RNA suppresses laser-induced choroidal neovascularization. Difference in ischemic regulation of vascular endothelial growth factor and pigment epithelium-derived factor in Brown Norway and Sprague Dawley rats contributing to different susceptibilities to retinal neovascularization.
Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth, and pathological angiogenesis. DNA damage response and autophagy in the degeneration of retinal pigment epithelial cells – implications for age-related macular degeneration (AMD). Anti-angiogenic effects of mammalian target of rapamycin inhibitors in a mouse model of oxygen-induced retinopathy.
Stabilization of HIF-2alpha through redox regulation of mTORC2 activation and mRNA translation initiation.
