Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imaging and Treating Choroidal Neovascularization

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV

Effect of Adding Dexamethasone to Continued Ranibizumab Treatment in Patients With Persistent Diabetic Macular Edema

In this phase 2 randomized clinical trial, the Diabetic Retinopathy Clinical Research Network compares continued ranibizumab treatment alone vs ranibizumab plus dexamethasone in patients with persistent diabetic macular edema

Bioimaging with NP-angiography showing GFP expression using the Topcon camera with fluorescein angiography filter settings.

<p>Late phase FAs (A and D) show the CNV lesions prior to injection of NP. Autofluorescent images taken prior to injection of NP reveal minimal background fluorescence of the CNV lesions (B and E). Injection of targeted NP carrying a GFP plasmid (NP-GFPg) causes increased fluorescence of the CNV lesions from GFP expression (C) whereas non-targeted NP carrying a GFP plasmid (ntNP-GFPg) does not cause any increase in the intensity of fluorescence of the CNV over background autofluorescence (F).</p

Evaluation of endothelial cell apoptosis with TUNEL staining in frozen sections.

<p>Quantification of TUNEL positive cells showed significantly more TUNEL(+) cells/lesion (A) and TUNEL (+) cells/mm² (B) with treatment of NP-ATPμ-Raf compared to the control group on day 3 and 5 after laser injury. There was a statistically significant reduction of CNV size noted on day 7(C). Double-immunofluorescent staining of frozen sections (<i>x20</i>) obtained at 3, 5 and 7 days after laser photocoagulation for the endothelial cell marker CD31 and TUNEL stain (D). *P<0.01. Data are expressed as the mean ± SE.</p

<i>In vivo</i> evaluation of CNV utilizing SD-OCT.

<p>Quantification of CNV size using SD-OCT (A) reveals a decrease in CNV size, reaching statistical significance on day 7 (Mann- Whitney U test, p = 0.001) in the NP-ATPμ-Raf treated group compared to the control group. A hyper-reflective subretinal lesion is seen as delineated by the red dotted line (B). This lesion corresponds to the hyporeflective area on fundus reconstruction (red dotted circle, C). *P<0.01. Data are expressed as the mean ± SE.</p

Late phase fluorescein angiography (FA) and choroidal flatmounts (<i>x10</i>) two weeks after laser photocoagulation.

<p>Representative lesions are from the control group (A–D) and the NP-ATPμ-Raf treated group (E and F). Group (A) received no treatment; (B) received intravenous injection of non-targeted NP containing ATPμ-Raf on days 1, 3, and 5 after laser CNV creation; (C) received intravenous injection of α_νβ₃ targeted-NP without ATPμ-Raf gene on days 1,3, and 5; (D) received injection of ATPμ-Raf gene without NP on days 1, 3, and 5; (E) received injection of α_νβ₃ targeted-NP containing ATPμ-Raf (NP-ATPμ-Raf) on days 1, 3, and 5; and (F) received injection of NP-ATPμ-Raf on days 3, 5, and 7. NP-ATPμ-Raf treated groups (E and F) had significantly lower grade CNV lesions on FA grading and smaller CNV size compared to the control group (A–D). No statistically significant difference in size was noted between the control groups A–D. Quantification of the CNV size on choroidal flat mounts is shown in (G). *P<0.01. Data are expressed as the mean ± SE.</p

Choroidal flatmounts showing accumulation of rhodamine labeled NP and expression of GFP plasmid in the CNV.

<p>The CNV lesions are delineated by arrowheads in bright field images with false blue color (A and E). FITC-filtered images highlight the GFP expression one day after systemic injection of Rd-NP-GFPg (B) whereas non-targeted NP (Rd-ntNP-GFPg) does not induce GFP expression in CNV (F). Cy3-filtered images highlight that rhodamine-labeled NP (Rd-NP-GFPg) accumulates in the CNV (C), while rhodamine-labeled non-targeted NP (Rd-ntNP-GFPg) does not (G). Some particles can be visualized circulating in the choroidal vessels. Overlay of images A–C is presented in panel D and overlay of E–G is shown in H.</p