Inhibition of choroidal neovascularization in a nonhuman primate model by intravitreal administration of an AAV2 vector expressing a novel anti-VEGF molecule.

Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV). A dose-dependent and persistent expression of sFLT01 was observed by collecting samples of aqueous humor at different time points over 5 months. The location of transduction as elucidated by in situ hybridization was in the transitional epithelial cells of the pars plana and in retinal ganglion cells. AAV2-sFLT01 was able to effectively inhibit laser-induced CNV in a dose-dependent manner as determined by comparing the number of leaking CNV lesions in the treated versus control eyes using fluorescein angiography. Our data suggest that intravitreal delivery of AAV2-sFLT01 may be an effective long-term treatment for diseases caused by ocular neovascularization.

Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing.

Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved ∼1-6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in ∼1-2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.

Gene Therapies for Neovascular Age-Related Macular Degeneration.

Pathological neovascularization is a key component of the neovascular form (also known as the wet form) of age-related macular degeneration (AMD) and proliferative diabetic retinopathy. Several preclinical studies have shown that antiangiogenesis strategies are effective for treating neovascular AMD in animal models. Vascular endothelial growth factor (VEGF) is one of the main inducers of ocular neovascularization, and several clinical trials have shown the benefits of neutralizing VEGF in patients with neovascular AMD or diabetic macular edema. In this review, we summarize several preclinical and early-stage clinical trials with intraocular gene therapies, which have the potential to reduce or eliminate the repeated intravitreal injections that are currently required for the treatment of neovascular AMD.

Aurintricarboxylic acid increases yield of HSV-1 vectors.

Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adeno-associated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid. In our search for compounds that would increase HSV-1 yield, we investigated another anti-inflammatory compound, aurintricarboxylic acid (ATA). Although ATA has been previously shown to have antiviral effects, we find that low (micromolar) concentrations of ATA increased HSV-1 vector production yields. Our results showing the use of ATA to increase HSV-1 titers have important implications for the production of certain HSV-1 vectors as well as recombinant AAV vectors.

AAV5-mediated sFLT01 gene therapy arrests retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice.

To test the effects of adeno-associated virus encoding sFLT01 (AAV5.sFLT01) on the retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice, a model for age-related macular degeneration (AMD), AAV5.sFLT01 was injected into the subretinal space of the right eyes and the left eyes served as controls. Histology found no retinal toxicity due to the treatment after 3 months. The treated eyes showed lesion arrest compared with lesion progression in the left eyes by fundus monitoring monthly and histological evaluation 3 months after treatment. Retinal ultrastructure showed fewer lipofuscin and better preserved photoreceptors after the treatment. A2E, a major component of lipofuscin, was lower in the treated eyes than in the control eyes. Molecular analysis showed that AAV5.sFLT01 lowered retinal extracellular signal-regulated kinase (ERK) phosphorylation and inducible nitric oxide synthetase expression, which suggested the involvement of reactive nitrogen species in the retinal lesions of Ccl2(-/-)/Cx3cr1(-/-). We concluded that local delivery of AAV5.sFLT01 can stabilize retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice. The findings provide further support for the potential beneficial effects of sFLT01 gene therapy for age-related macular degeneration.

sFLT01: a novel fusion protein with antiangiogenic activity.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG(1) through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non-small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology.