Abstract
Purpose: :
Angiogenesis plays a key role in the pathophysiology of many ocular conditions such as age-related macular degeneration (AMD). Angiogenesis is also important in cancer growth in order to provide nutrients and metabolic support to tumors. VEGFR2 is an important receptor involved in neovascular signaling. Because of alternative splicing, it is present in both membrane-bound (mVEGFR2) and soluble (sVEGFR2) forms. mVEGFR2 is pro-angiogenic, while sVEGFR2 is anti-angiogenic. We have developed a morpholino-based gene therapy technique that shifts expression from mVEGFR2 to sVEGFR2. Here we present the success of this technique in treating a murine model of choroidal neovascularization (CNV). In a follow-up experiment we show that this treatment is able to reduce tumor growth in a xenograft tumor model.
Methods: :
CNV was induced in C57 mice using laser photocoagulation. On day 1 and day 4 after photocoagulation, either control morpholino, sVEGFR2-inducing morpholino, or DBPS was injected intravitreously. On day 7, CNV volumes were measured by confocal microscopy.NMRI nu/nu mice received subcutaneous injections of HCT 116 colon cancer cells for the xenograft tumor experiments. One week after the injection of cells, treatment began with control morpholino, sVEGFR2-inducing morpholino, or HBSS. Tumors were injected with treatment twice weekly for a period of three weeks. Tumor size was measured twice weekly using calipers. A subset of tumors was harvested after the first treatment injection. RNA from these tumors was extracted, and RT-PCR was performed to evaluate levels of both mVEGFR2 and sVEGFR2.
Results: :
There was a 50% reduction of CNV in the mice treated with the sVEGFR2-inducing morpholino compared to the mice treated with control morpholino (n=11-17).There was a 53% reduction in tumor volume in the tumors treated with the sVEGFR2-inducing morpholino compared to the mice treated with control morpholino (n=5). Additionally, the sVEGFR2-inducing morpholino showed a reduced expression of mVEGFR2 and increased expression of sVEGFR2 as confirmed by RT-PCR.
Conclusions: :
Anti-sense morpholinos targeting the exon13-intron13 junction of VEGFR2 shift expression from mVEGFR2 to sVEGFR2. Here we have shown that this results in a decrease in CNV volume in a laser CNV model. We have also demonstrated that increased sVEGFR2 can reduce tumor growth in a xenograft model. This is an interesting example of a therapy developed for ocular diseases being used for cancer therapy. The splicing shift described in this study shows promise as a therapeutic method for neovascular-related disorders.
Keywords: neovascularization • vascular endothelial growth factor • choroid: neovascularization