title: Enhancement of rAAV2-mediated transgene expression in retina cells in vitro and in vivo by coadministration of low-dose chemotherapeutic drugs.
abstract: OBJECTIVE
  Recombinant adeno-associated viral vector serotype 2 (rAAV2) has been used with success to deliver retina-targeted gene therapeutics in retinal degeneration. However, one of the major limitations of this approach is the vector's low transduction efficiency. This study is designed to increase AAV2 transduction efficiency in vitro and in vivo.
  
  
  METHODS
  Green fluorescence protein (GFP) or luciferase reporter gene-carried rAAV2 vectors were applied to cultured human RPE cells (ARPE-19) or animal eyes with or without chemotherapeutic agents. GFP transduction efficiency was evaluated by image, flow cytometry analysis, and Western blot. The ciliary neurotrophic factor (rAAV2-CNTF)-carried AAV2 vector was coinjected to subretinal space with or without chemotherapeutic agent. The therapeutic efficacy was evaluated by counting numbers of remaining photoreceptors in retina sections of treated or untreated eyes.
  
  
  RESULTS
  Coadministration of 0.1 μg/mL doxorubicin (DXR), 0.14 μg/mL cytarabine (Ara-C), 1 μg/mL etoposide (VP-16), or 20 μg/mL cisplatin (DDP) significantly increased rAAV2-mediated GFP and/or luciferase expression in cultured hRPE cells without any detectable toxicity. Pretreatment with DXR for 24 h prior to infection was most effective in enhancing rAAV2 transgene expression in hRPE cells. In addition, subretinal coinjection of rAAV2-CMV-ciliary neurotrophic factor (rAAV2-CNTF) and DXR into the eyes of rats with inherited retinal degeneration resulted in an approximately 2-fold increase in photoreceptor layer thickness and cellular density of the outer nuclear layer (ONL) compared to rAAV2-CNTF alone, reflecting a pronounced protection effect mediated by the enhanced expression of CNTF.
  
  
  CONCLUSIONS
  The method described here to improve rAAV2-based gene delivery is simple and feasible without any detectable toxicity. This strategy might be therapeutically exploited in the gene therapy of degenerative retinal diseases.
authors.affiliation: Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
authors.forename: Shenghai
authors.initials: S
authors.lastname: Zhang
authors.forename: Jihong
authors.initials: J
authors.lastname: Wu
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Wu
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Tian
authors.forename: Miaoying
authors.initials: M
authors.lastname: Yi
authors.forename: Xinjian
authors.initials: X
authors.lastname: Liu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Dong
authors.forename: Frank
authors.initials: F
authors.lastname: Wolf
authors.forename: Chuanyuan
authors.initials: C
authors.lastname: Li
authors.forename: Qian
authors.initials: Q
authors.lastname: Huang
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D000229:Dependovirus / Q000235:genetics*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011922:Rats, Mutant Strains; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D019076:Transgenes
chemical_list: D000970:Antineoplastic Agents; D020934:Ciliary Neurotrophic Factor; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases