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Updated: May 20, 2025
Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
Leveraging AAV1-Rac1T17N to prevent experimental proliferative vitreoretinopathy
Duo Li1,2, Minli Linghu1, Jisen Tang1,2
1Department of Ophthalmology, The First Affiliated Hospital of Hainan Medical University, Haikou, 570102, China.
Background:
Platelet-derived growth factor receptor β (PDGFRβ) is the principal PDGFR isoform in retinal pigment epithelial (RPE) cells from the epiretinal membranes of patients with proliferative vitreoretinopathy (PVR). Ras-related C3 Botulinum toxin substrate 1 (Rac1), a member of the Rho family, is a crucial factor in the cell migration and contraction processes that are inherent to the pathogenesis of PVR. The mutants Rac1T17N and Rac1Q61L can block and promote Rac1 activation, respectively. The major objective of this research was to ascertain whether PDGFRβ mediates vitreous-induced Rac1 activation and whether Rac1T17N could be leveraged for the prevention of PVR pathogenesis in a rabbit model.
Methods:
A pull-down assay was used to examine GTP Rac1 levels, which are indicative of Rac1 activation, and western blotting was used to assess cellular protein expression. A CCK8 assay, a wound healing assay, a transwell invasion assay and a collagen contraction assay were employed to analyze cell proliferation, migration, invasion and contraction capacity, respectively. A PVR model was created by injecting platelet-rich plasma and human retinal pigment epithelial cells (ARPE-19) into the vitreous cavities of rabbits, and this model was used to evaluate the severity of PVR impacted by intravitreally injected ARPE-19 cells transduced with adeno-associated virus (AAV)1-Rac1T17N or Rac1Q61L. PVR grade was evaluated by a double-blinded investigator according to the Fastenberg classification; in addition, ultrasound and histological analyses were performed to assess PVR severity.
Results:
Vitreous-induced GTP Rac1 is mediated by PDGFRβ. There was a significant decrease in vitreous-induced GTP Rac1 in ARPE-19 cells transduced with AAV1-Rac1T17N compared with those transduced with AAV1-GFP. In addition, the suppression of GTP Rac1 production in human RPE cells by transduction with AAV1-Rac1T17N inhibited vitreous-induced proliferation, migration, invasion, and contractility. Importantly, the results of the animal experiments indicated that although there was a significant increase in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1Q61L, there was a significant decrease in PVR potential in rabbits intravitreally injected with ARPE-19 cells infected with AAV1-Rac1T17N (P < 0.01).
Conclusions:
AAV1-Rac1T17N has great potential for PVR therapy.
