AAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization

Abstract

Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.

Inhibition of Experimental Choroidal Neovascularization by a Novel Peptide Derived from Calreticulin Anti-Angiogenic Domain

“…Our previous studies have demonstrated that the gene delivery of CAD and its derived fragment (CAD112) can attenuate the development of choroidal and retinal neovascularization in rodent models of laser-induced CNV and oxygen-induced retinopathy [ 18 ]. However, adverse effects associated with prolonged expression of vascular targeting proteins by gene delivery may have unwanted side effects, including retinal vascular toxicity.…”

“…One day after laser-induced CNV induction, rats were anesthetized with a combination of xylocaine (0.15 mL/kg body weight) and ketamine (50 mg/kg body weight). Intravitreal injection was performed under a surgical microscope as previously described [ 18 ]. After a small puncture through the conjunctiva and sclera was created using a 30 gauge needle, a 32 gauge blunt needle connected to a 10-μL Hamilton syringe was inserted into the vitreous and 5 μL of DPBS suspension containing Lucentis ® (ranibizumab, 50 μg; Novartis, Basel, Switzerland), CAD27 (10 and 20 μg), or vehicle (DPBS) was injected into one eye of each rat using a UMP3–2 Ultra Micro Pump (World Precision Instruments, Sarasota, FL, USA) at a rate of 100 nL/s.…”

Inhibition of Experimental Choroidal Neovascularization by a Novel Peptide Derived from Calreticulin Anti-Angiogenic Domain

“…Our previous studies have demonstrated that gene delivery of CAD and its derived fragment (CAD112) can attenuate the development of choroidal and retinal neovascularization in rodent models of laser-induced CNV and oxygen-induced retinopathy [18]. However, adverse effects associated with prolonged expression of vascular targeting proteins by gene delivery may have unwanted side effects, including retinal vascular toxicity.…”

“…One day after laser-induced CNV induction, rats were anesthetized with a combination of xylocaine (0.15 mL/kg body weight) and ketamine (50 mg/kg body weight). Intravitreal injection was performed under a surgical microscope as previously described [18] .…”

Updates on Gene Therapy for Diabetic Retinopathy

“…In addition, retinal vascular architecture has been partly preserved in those mice injected with AAV expressing endostatin [17]. Retinal transduction of AAV encoding other endogenous angiogenesis inhibitors such as tissue inhibitor metalloproteinase-3 or CAD has also been found to reduce retinal neovascularization in OIR mice [16,27].…”