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Lentivirus-mediated RNA interference of vascular endothelial growth factor in monkey eyes with iris neovascularization.

Yuan MK, Tao Y, Yu WZ, Kai W, Jiang YR - Mol. Vis. (2010)

Bottom Line: At 50 days after laser treatment, only a slight ectropion uvea was found.However, in the other eyes, obvious INV or hyphema was observed.Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, People's Hospital, Peking University, Beijing, China.

ABSTRACT

Purpose: To explore the in vivo anti-angiogenesis effects resulting from lentivirus-mediated RNAi of vascular endothelial growth factor (VEGF) in monkeys with iris neovascularization (INV).

Methods: Five specific recombinant lentiviral vectors for RNA interference, targeting Macaca mulatta VEGFA, were designed and the one with best knock down efficacy (LV-GFP-VEGFi1) in H1299 cells and RF/6A cells was selected by real-time PCR for in vivo use. A laser-induced retinal vein occlusion model was established in one eye of seven cynomolgus monkeys. In monkeys number 1, 3, and 5 (Group 1), the virus (1x10(8) particles) was intravitreally injected into the preretinal space of the animal's eye immediately after laser coagulation; and in monkeys number 2, 4, and 6 (Group 2), the virus (1x10(8) particles) was injected at 10 days after laser coagulation. In monkey number 7, a blank control injection was performed. In monkeys number 1 and 2, virus without RNAi sequence was used; in monkeys number 3 and 4, virus with nonspecific RNAi sequence was used; and in monkeys 5 and 6, LV-GFP-VEGFi1 was used.

Results: In monkey number 5, at 23 days after laser treatment, no obvious INV was observed, while fluorescein angiography of the iris revealed high fluorescence at the margin of pupil and point posterior synechiae. At 50 days after laser treatment, only a slight ectropion uvea was found. However, in the other eyes, obvious INV or hyphema was observed. The densities of new iridic vessels all significantly varied: between monkey number 5 and number 3 (36.01+/-4.49/mm(2) versus 48.68+/-9.30/mm(2), p=0.025), between monkey number 3 and monkey number 7 (48.68+/-9.30/mm(2) versus 74.38+/-9.23/mm(2), p=0.002), and between monkey number 5 and number 7 (36.01+/-4.49/mm(2) versus 74.38+/-9.23/mm(2), p<0.001).

Conclusions: Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

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Related in: MedlinePlus

Construction of a lentivirus-mediated shRNA of Macaca mulatta VEGFA.
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f1: Construction of a lentivirus-mediated shRNA of Macaca mulatta VEGFA.

Mentions: shRNA of Macaca mulatta VEGFA lentivirus gene transfer vector encoding green fluorescent protein (GFP) sequence was constructed by Genechem Co., Ltd, Shanghai, China (Figure 1). Five targeting sequences of the shRNA were designed as follows: 5′- AAT GCA GAC CAA AGA AAG ATA-3′ (VEGFi1), 5′-AGG GCA GAA TCA TCA CGA AGT-3′ (VEGFi2), 5′-CGA ACG TAC TTG CAG ATG TGA-3′ (VEGFi3), 5′-GAC GTG TAA ATG TTC CTG CAA-3′ (VEGFi4), and 5′-ATG CGG ATC AAA CCT CAC CAA-3′ (VEGFi5; GenBank XM_001089925). The lentivirus-GFP (LV-GFP) which included the GFP gene and did not include the VEGFA interference sequence served as negative control, and the target sequence, VEGFiCON (5′-TTC TCC GAA CGT GTC ACG T-3′), was designed with a randomly chosen nonsense sequence to serve as an additional control. The shRNA was confirmed by sequencing.


Lentivirus-mediated RNA interference of vascular endothelial growth factor in monkey eyes with iris neovascularization.

Yuan MK, Tao Y, Yu WZ, Kai W, Jiang YR - Mol. Vis. (2010)

Construction of a lentivirus-mediated shRNA of Macaca mulatta VEGFA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927369&req=5

f1: Construction of a lentivirus-mediated shRNA of Macaca mulatta VEGFA.
Mentions: shRNA of Macaca mulatta VEGFA lentivirus gene transfer vector encoding green fluorescent protein (GFP) sequence was constructed by Genechem Co., Ltd, Shanghai, China (Figure 1). Five targeting sequences of the shRNA were designed as follows: 5′- AAT GCA GAC CAA AGA AAG ATA-3′ (VEGFi1), 5′-AGG GCA GAA TCA TCA CGA AGT-3′ (VEGFi2), 5′-CGA ACG TAC TTG CAG ATG TGA-3′ (VEGFi3), 5′-GAC GTG TAA ATG TTC CTG CAA-3′ (VEGFi4), and 5′-ATG CGG ATC AAA CCT CAC CAA-3′ (VEGFi5; GenBank XM_001089925). The lentivirus-GFP (LV-GFP) which included the GFP gene and did not include the VEGFA interference sequence served as negative control, and the target sequence, VEGFiCON (5′-TTC TCC GAA CGT GTC ACG T-3′), was designed with a randomly chosen nonsense sequence to serve as an additional control. The shRNA was confirmed by sequencing.

Bottom Line: At 50 days after laser treatment, only a slight ectropion uvea was found.However, in the other eyes, obvious INV or hyphema was observed.Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, People's Hospital, Peking University, Beijing, China.

ABSTRACT

Purpose: To explore the in vivo anti-angiogenesis effects resulting from lentivirus-mediated RNAi of vascular endothelial growth factor (VEGF) in monkeys with iris neovascularization (INV).

Methods: Five specific recombinant lentiviral vectors for RNA interference, targeting Macaca mulatta VEGFA, were designed and the one with best knock down efficacy (LV-GFP-VEGFi1) in H1299 cells and RF/6A cells was selected by real-time PCR for in vivo use. A laser-induced retinal vein occlusion model was established in one eye of seven cynomolgus monkeys. In monkeys number 1, 3, and 5 (Group 1), the virus (1x10(8) particles) was intravitreally injected into the preretinal space of the animal's eye immediately after laser coagulation; and in monkeys number 2, 4, and 6 (Group 2), the virus (1x10(8) particles) was injected at 10 days after laser coagulation. In monkey number 7, a blank control injection was performed. In monkeys number 1 and 2, virus without RNAi sequence was used; in monkeys number 3 and 4, virus with nonspecific RNAi sequence was used; and in monkeys 5 and 6, LV-GFP-VEGFi1 was used.

Results: In monkey number 5, at 23 days after laser treatment, no obvious INV was observed, while fluorescein angiography of the iris revealed high fluorescence at the margin of pupil and point posterior synechiae. At 50 days after laser treatment, only a slight ectropion uvea was found. However, in the other eyes, obvious INV or hyphema was observed. The densities of new iridic vessels all significantly varied: between monkey number 5 and number 3 (36.01+/-4.49/mm(2) versus 48.68+/-9.30/mm(2), p=0.025), between monkey number 3 and monkey number 7 (48.68+/-9.30/mm(2) versus 74.38+/-9.23/mm(2), p=0.002), and between monkey number 5 and number 7 (36.01+/-4.49/mm(2) versus 74.38+/-9.23/mm(2), p<0.001).

Conclusions: Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

Show MeSH
Related in: MedlinePlus