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Biochemistry and Molecular Biology  |   May 2012
Enhancement of rAAV2-Mediated Transgene Expression in Retina Cells In Vitro and In Vivo by Coadministration of Low-Dose Chemotherapeutic Drugs
Author Affiliations & Notes
  • Shenghai Zhang
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Jihong Wu
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Xiaobing Wu
    State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China;
  • Ping Xu
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Yuhua Tian
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Miaoying Yi
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Xinjian Liu
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Xiaoyan Dong
    State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China;
  • Frank Wolf
    Department of Radiation Oncology, Medical University of Innsbruck, Innsbruck, Austria; and
  • Chuanyuan Li
    Department of Dermatology, Medical Center, Duke University, Durham, North Carolina.
  • Qian Huang
    Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China;
  • Corresponding authors: Chuanyuan Li, Department of Dermatology, Medical Center, Duke University, Durham, NC 27710; Office Tel: (919) 613-8754; chuan.li@duke.edu; and Qian Huang, Experimental Research Center, First People's Hospital, School of Medicine, Shanghai Jiaotong University, 85 Wujin Road, Shanghai 200080 China; qhuang@sjtu.edu.cn.  
Investigative Ophthalmology & Visual Science May 2012, Vol.53, 2675-2684. doi:10.1167/iovs.11-8856
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      Shenghai Zhang, Jihong Wu, Xiaobing Wu, Ping Xu, Yuhua Tian, Miaoying Yi, Xinjian Liu, Xiaoyan Dong, Frank Wolf, Chuanyuan Li, Qian Huang; Enhancement of rAAV2-Mediated Transgene Expression in Retina Cells In Vitro and In Vivo by Coadministration of Low-Dose Chemotherapeutic Drugs. Invest. Ophthalmol. Vis. Sci. 2012;53(6):2675-2684. doi: 10.1167/iovs.11-8856.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: 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.

Introduction
Retinal diseases such as age-related macular degeneration, diabetic retinopathy, or retinopathy of prematurity are common eye diseases associated with vasculopathy of small retinal vessels and can progress to total blindness if not treated. 13 Treatment options include surgery, laser therapy, and photodynamic therapy. These treatment options often fail to prevent recurrence of the disease since they do not eradicate angiogenic stimuli that are involved in disease recurrence. 4,5 Therefore, persistent targeted expression of angiogenic inhibitors is a promising approach currently being investigated by many groups. 6,7 Intravitreal injection of the vascular endothelial growth factor (VEGF)-A antibody ranibizumab has been successfully used to inhibit retinal angiogenesis. 4,5 However, the therapeutic effect is of short duration, necessitating frequent injections. Therefore, longer-lasting antiangiogenic therapies, for example by delivering antiangiogenic transgenes that are persistently expressed in retinal cells, are needed. 6,7  
The retina is an ideal candidate for gene therapy since it is easily accessible to local injection and allows simple functional assessment using visual tests. In addition, the eye–blood barrier limits systemic diffusion of viral vectors, ensuring safe application and providing an immunologically privileged environment. 
Inherited forms of retinal dystrophia are caused by a number of genetic defects ultimately leading to photoreceptor cell death. Since targeting defective genes is difficult due to the large number of implicated genes, therapeutic strategies are aiming to deliver holistic survival factors to promote photoreceptor survival globally. 812 However, application of these strategies requires an efficient delivery vector. 
Recombinant adeno-associated virus vector (rAAV) is the most widely used and most promising gene delivery vector to the retina since it exhibits low immunogenicity and induces minimal cytotoxic response. 1315 In addition, wild-type AAV virus can stably integrate site-specifically into the host genome at a specific locus of human chromosome 19 in a latent infection. 16 Recombinant AAV type 2 (rAAV2) has been shown to carry transgenes up to 4.7 kb or bigger and to achieve long-term expression of transgenes in episome form. 1719 Recombinant AAV2 has been successfully used to mediate efficient and prolonged transgene expression in a number of tissues, and has been therapeutically used in clinical trails for human genetic retinal disorders. 2022  
However, relatively low transduction efficiency of rAAV2 prevents the vector from seeing wide application in the clinic. Much effort has been undertaken to increase rAAV2 transduction efficacy, and promising results have been reported for several reagents and treatments, such as hydroxyurea, UV or ionizing irradiation, histone deacetylase (HDAC) inhibitors, and topoisomerase inhibitors. 2328 The underlying mechanism for most of these treatments seems to be an enhanced viral entry or conversion of AAV single-stranded DNA into double-stranded forms, which is the rate-limiting step in rAAV2 transduction. 2931 In contrast, HDAC inhibitors have been shown to enhance transduction efficacy by inducing increased acetylation of histone-associated chromatin of the rAAV genome. 24  
In searching for alternative strategies to increase rAAV2 transduction, the present authors hypothesized that classic chemotherapeutic agents widely used in cancer treatment might improve rAAV2-mediated transgene expression and have a beneficial toxicity profile compared to the above-mentioned methods. 
Materials and Methods
Viral Vectors and Cells
Recombinant adeno-associated viral vectors rAAV2-CMV-EGFP (referred to as rAAV2-GFP), rAAV2-CMV-luciferase (rAAV2-Luc), and rAAV2-CMV-ciliary neurotrophic factor (rAAV2-CNTF) were purchased from Vector Gene Technology Company Limited (Beijing, China). Human retinal pigment epithelium (RPE) cell line ARPE-19 (ATCC, Rockville, MD) was grown in Dulbecco's modified Eagle's medium/F12 (Gibco, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS) at 37°C, 5% CO2
In Vitro Infection and Transgene Expression
For green fluorescence protein (GFP) expression image analysis, 1 × 1. 5 APRE-19 cells were seeded into 24-well plates 24 h prior to infection and infected with rAAV2-GFP at 1 × 1. 2 , 1 × 1. 3 , and 1 × 1. 4 vector genomes per cell (vg/cell) as well as different concentrations of DXR, Ara-C, VP-16, or DDP for 72 h, except as otherwise indicated. Uninfected and rAAV2-EGFP–only infected cells were used as controls. GFP expression was imaged using an inverted fluorescence microscope (Axio 100; Zeiss, Jena, Germany) at 24 h intervals postinfection. For quantitative analysis of GFP expression, fluorescence-activated cell scanning (FACS; EPICS XL; Beckman Coulter Company, Miami, FL) was conducted. All experiments were repeated at least three times. 
Real-time PCR for mRNA Analysis
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The single-stranded cDNA was reverse transcribed using a Superscript III first-strand synthesis system (Invitrogen). A total of 100 ng cDNA was amplified in a 50-μL reaction mixture. The mRNA levels of GFP, fibroblast growth factor receptor (FGFR), heparin sulfate proteoglycan (HSPG), or alpha ν integrin were quantified by real-time PCR (MJ Research, Waltham, MA), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as internal control. The relative expression of target genes was normalized with a calibrator (hRPE cells infected with rAAV2-EGFP only). The final difference, expressed as N-fold difference in target gene expression relative to GAPDH and the calibrator, was determined by the following formula: Ntarget = 2−ΔΔCt
Determination of Transgene Copy Number
ARPE-19 cells were infected as described above, and total DNA was extracted using a DNA extraction kit (Invitrogen). One hundred nanograms of deoxyribonucleic acid (DNA) was amplified in a 50-μL reaction mixture using the Platinum SYBR Green qPCR Supermix UDG kit (Invitrogen). The GFP copy numbers were determined by real-time PCR and presented as N-fold differences relative to the calibrator as described above. The GAPDH copy number was used as an internal control. 
Subretinal Injection
Normal adult Sprague-Dawley (SD) rats (180–200 g), Balb/c mice (18–20 g), and Royal College of Surgeons (RCS) rats (3 weeks old), an animal model for inherited retinal degeneration. 32 were obtained from Shanghai Laboratory Animal Center (SLAC). All animals were treated, maintained, and sacrificed in accordance with the policies specified in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines approved by national and local institutions. Surgical procedures and postsurgery care were performed as described previously. 33 Eyes of SD rats, Balb/c mice, and RCS rats were injected with rAAV2-EGFP, rAAV2-Luc, or rAAV2-CNTF alone at a dose of 5 × 1. 8 ∼ 1 × 1. 9 vg/eye with or without chemotherapeutic drugs. As a negative control, eyes were injected with PBS only. 
Fluorescence and Bioluminescence Imaging In Vivo
GFP fluorescence in the ocular fundus of living SD rats was monitored and imaged using a fluorescence stereoscope (Leica MZFLIII; Leica, Jena, Germany). ImagePro Plus software (Media Cybernetics, Silver Spring, MD) was used for image analysis and quantification of GFP intensity. Bioluminescence imaging of luciferase activity in the eyes of Balb/c mice was performed after intraperitoneal injection of D-luciferin using an in vivo imaging system (Roper Scientific, Trenton, NJ). 
Tissue Processing and H&E Staining
RCS rats were sacrificed with an overdose of intraperitoneally injected 10% chloral hydrate 8 weeks after subretinal injection. After enucleation, eyes were fixed in 10% formaldehyde solution, embedded in paraffin, and sliced into 5-μm sections. Sections were stained with hematoxylin and eosin (H&E) and imaged using a light microscope (Axioplan2 imaging; Zeiss, Jena, Germany). 
Morphometric Analysis of Photoreceptor Layers in RCS Rats
Morphometric analysis of the outer nuclear layer (ONL) in the retina was performed using a Zeiss Axioplan2 microscope and ImagePro Plus software. Measurements were performed in both the temporal and nasal halves of the retina as described previously. Three predefined regions (300 μm from the ora serrata and optic disc) were selected in each hemisphere using a 5× objective, and measurements for ONL thickness were taken for each region in five consecutive sections using a 40× objective. In addition, the rows of nuclei in the ONL were counted in multiple consecutive sections. P values were calculated using the paired Student's t-test. 
Western Blot Analysis
APRE cells and carefully dissected retinas were incubated with lysis buffer in Eppendorf tubes on ice for 15 minutes. Western blot analysis was performed as described previously. 33 Primary mouse monoclonal anti-GFP antibody (MBL, 1:10,000) and goat polyclonal anti-CNTF antibody (1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary peroxidase-conjugated anti-mouse and anti-goat antibodies were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). 
Results
Addition of Chemotherapeutic Drugs during Infection Increases rAAV2-Mediated GFP Expression in ARPE-19 Cells
AAV2-EGFP was used quantify rAAV2-mediated gene expression, which allowed monitoring of GFP expression levels using fluorescence microscopy. Human ARPE-19 cells were infected with rAAV2-EGFP alone or in the presence of one of the following cytotoxic drugs: DXR at a final concentration of 0.1 μg/mL, Ara-C at 0.14 μg/mL, VP-16 at 1 μg/mL, or DDP at 20 μg/mL. GFP expression was quantified 7 days after infection using a Zeiss Axio 100 fluorescence microscope. As shown in Figure 1A, control cells infected with rAAV2-EGFP alone exhibited very dim GFP fluorescence (upper panel). In contrast, treatment with DXR, Ara-C, and VP-16 dramatically increased the percentage of GFP-positive cells, as well as the overall GFP intensity in individual cells. On visual inspection, cells seemed healthy without apparent morphological changes or apoptotic features. All drugs seemed to enhance the transduction rate, that is, the percentage of GFP-positive cells, by about the same margin, while DXR, Ara-C, and VP-16 induced higher expression levels in individual cells reflected by a stronger GFP intensity. To verify this observation, the percentage and fluorescence intensity of GFP-positive cells were quantified by FACS analysis on day 4 and day 7 postinfection (Figs. 1B, 1C). Consistent with these observations, all chemotherapeutic drugs efficiently increased both the percentage of GFP-positive cells and individual GFP intensity (Figs. 1B, 1C). DDP was least effective in enhancing expression levels on a single-cell basis while maintaining the same rate of infection (Fig. 1A, 7 days postinfection). 
Figure 1.
 
Chemotherapeutic drugs increase rAAV2-delivered GFP expression in human retinal pigmented epithelium cells. (A) Representative fluorescence (upper panel) and bright field (lower panel) images of ARPE-19 cells after infection with rAAV2-EGFP alone (a, b), or coadministration of 0.1 μg/mL DXR (c, d), 0.14 μg/mL Ara-C (e, f), 1 μg/mL VP-16 (g, h), or 20 μg/mL DDP (i, j). Imaging was performed 7 days postinfection. (B) FACS analysis of ARPE-19 cells 7 days after infection with rAAV2-EGFP at a viral titer of either 1. 2 , 1. 3 , or 1. 4 vg/cell with or without the presence of chemotherapeutic drugs as indicated. Percentage of GFP-positive cells (B) and mean intensity of GFP fluorescence (C) are plotted. Data are presented as mean ± SD. N ≥ 3. BF, bright field.
Figure 1.
 
Chemotherapeutic drugs increase rAAV2-delivered GFP expression in human retinal pigmented epithelium cells. (A) Representative fluorescence (upper panel) and bright field (lower panel) images of ARPE-19 cells after infection with rAAV2-EGFP alone (a, b), or coadministration of 0.1 μg/mL DXR (c, d), 0.14 μg/mL Ara-C (e, f), 1 μg/mL VP-16 (g, h), or 20 μg/mL DDP (i, j). Imaging was performed 7 days postinfection. (B) FACS analysis of ARPE-19 cells 7 days after infection with rAAV2-EGFP at a viral titer of either 1. 2 , 1. 3 , or 1. 4 vg/cell with or without the presence of chemotherapeutic drugs as indicated. Percentage of GFP-positive cells (B) and mean intensity of GFP fluorescence (C) are plotted. Data are presented as mean ± SD. N ≥ 3. BF, bright field.
Dose Effect and Time Course of Drug-Enhanced rAAV2-Mediated EGFP Expression
To characterize the dose dependency of chemotherapeutics on rAAV2-mediated expression levels, different concentrations of DXR (0.001–5 μg/mL), coadministered during infection of ARPE-19 cells with rAAV2-EGFP, were tested. Imaging was performed 7 days postinfection. As shown in Figure 2A, expression levels of GFP peaked at a DXR concentration of 0.1 μg/mL. Doses up to 2 μg/mL were well tolerated. At 5 μg/mL, cell growth was strongly inhibited and apoptosis induced as cells rounded up and detached from the surface, resulting in a disrupted subconfluent monolayer. 
Figure 2.
 
Optimization of doxorubicin dosage. (A) Representative fluorescence images of ARPE-19 cells 7 days after infection with rAAV2-EGFP in the presence of increasing amounts of DXR as indicated. (B, C) FACS analysis of APRE-19 cells at different time points after infection with rAAV2-EGFP (1. 4 vg/cell). The percentage of GFP-positive cells (B) as well as the mean overall GFP intensity (C) is shown for different concentrations of DXR present at the time of infection. Data are presented as mean ± SD. N ≥ 3.
Figure 2.
 
Optimization of doxorubicin dosage. (A) Representative fluorescence images of ARPE-19 cells 7 days after infection with rAAV2-EGFP in the presence of increasing amounts of DXR as indicated. (B, C) FACS analysis of APRE-19 cells at different time points after infection with rAAV2-EGFP (1. 4 vg/cell). The percentage of GFP-positive cells (B) as well as the mean overall GFP intensity (C) is shown for different concentrations of DXR present at the time of infection. Data are presented as mean ± SD. N ≥ 3.
To assess expression dynamics, GFP expression was monitored at different time points after infection. As shown in Figures 2B and 2C, both the percentage of GFP-positive cells and the overall GFP intensity increased over time. The effect was most pronounced at a DXR concentration of 0.1 μg/mL. Similar dynamics were observed with VP-16– and Ara-C (Fig. 3A)–treated cells. 
Figure 3.
 
Cytarabine (Ara-C) and etoposide (VP-16) increase AAV2-mediated transgene expression. (A) FACS analysis showing the percentage of GFP-positive cells and the mean GFP fluorescence intensity in APRE-19 cells that had been infected with rAAV2-EGFP (1. 4 vg/cell) in the presence of different concentrations of VP-16 (upper panel) and Ara-C (lower panel) at various time points. Data are presented as mean ± SD. N ≥ 3. (B) Luciferase activity (RL) of RPE cells infected with rAAV2-Luc alone or in the presence of different concentrations of VP-16. Luciferase activity was measured 3 days after infection. Data are presented as mean ± SD. N ≥ 3. (C) Representative Western blot of GFP expression in ARPE-19 cells 7 days after infection with rAAV2-EGFP (1. 4 vg/cell) with or without the presence of different concentrations of Ara-C. β-Actin was used as loading control.
Figure 3.
 
Cytarabine (Ara-C) and etoposide (VP-16) increase AAV2-mediated transgene expression. (A) FACS analysis showing the percentage of GFP-positive cells and the mean GFP fluorescence intensity in APRE-19 cells that had been infected with rAAV2-EGFP (1. 4 vg/cell) in the presence of different concentrations of VP-16 (upper panel) and Ara-C (lower panel) at various time points. Data are presented as mean ± SD. N ≥ 3. (B) Luciferase activity (RL) of RPE cells infected with rAAV2-Luc alone or in the presence of different concentrations of VP-16. Luciferase activity was measured 3 days after infection. Data are presented as mean ± SD. N ≥ 3. (C) Representative Western blot of GFP expression in ARPE-19 cells 7 days after infection with rAAV2-EGFP (1. 4 vg/cell) with or without the presence of different concentrations of Ara-C. β-Actin was used as loading control.
To demonstrate that drug-induced enhancement of expression is not limited to a particular target gene (GFP), an rAAV2 virus carrying an expression cassette for luciferase (rAAV2-Luc) was generated, and ARPE-19 cells were infected with either rAAV2-Luc alone or in the presence of different concentrations of DXR, Ara-C, DPP, and VP-16. Luciferase activity was assessed using a fluorescence spectrophotometer at 3 days postinfection. Consistent with the results obtained from the GFP expression experiments, all tested drugs efficiently increased luciferase activity in a dose-dependent manner. Exemplary results for VP-16–induced luciferase expression are shown in Figure 3B. Induction peaked at VP-16 concentrations of 1 μg/mL. At higher doses, luciferase activity dropped to control levels, presumably due to the cytotoxic effect of VP-16 at higher concentrations. 
To verify the results obtained from fluorescent imaging and flow cytometry analysis, GFP protein levels were tested by Western blot. As shown in Figure 3C, GFP expression was enhanced in a dose-dependent manner when Ara-C was added prior to infection. Similar results were obtained with VP-16, DXR, and DPP (data not shown). 
The results clearly showed that rAAV2 transduction efficiency, as well as expression levels of rAVV2-mediated genes in successfully transduced cells, could be significantly enhanced when chemotherapeutic drugs were present during the time of infection. The effect was most pronounced for DXR at doses of 0.1 μg/mL and peaked at 7 days postinfection. 
The Relative Time Point of Drug Administration Affects rAAV2 Transduction and Transgene Expression
To determine whether the relative time point of chemotherapeutic drug administration has any effect on rAAV2 transduction and rAAV2-delivered gene expression, cells were treated with DXR 24 h prior to, 24 h after, or simultaneously with rAAV2 infection. Percentage of GFP-positive cells and GFP intensity were analyzed 1, 2, 4, and 7 days after rAAV2 infection. Figures 4A and 4B show that pretreatment of cells with DXR for 24 h most effectively increased the percentage of GFP-positive cells, but the effect leveled out over time. Pretreatment with DXR achieved the best induction in GFP intensity at all tested time points. 
Figure 4.
 
Recombinant AAV2-mediated transgene expression is affected by the relative timing of drug administration. FACS analysis of APRE-19 cells at various time points after infection with rAAV2-EGFP and DXR 0.1, μg/mL, administered either 24 hours prior to, simultaneously with, or 24 hours after infection. Shown are the percentage of GF-positive cells (A) and the mean overall GFP fluorescence intensity (B) at different time points after infection as indicated.
Figure 4.
 
Recombinant AAV2-mediated transgene expression is affected by the relative timing of drug administration. FACS analysis of APRE-19 cells at various time points after infection with rAAV2-EGFP and DXR 0.1, μg/mL, administered either 24 hours prior to, simultaneously with, or 24 hours after infection. Shown are the percentage of GF-positive cells (A) and the mean overall GFP fluorescence intensity (B) at different time points after infection as indicated.
Chemotherapeutic Drugs Increase EGFP mRNA Copy Number, but Not mRNA Copy Numbers of Host Genes Associated with Viral Entry
To explore the mechanism of how chemotherapeutic drugs increased rAAV2-mediated gene expression, real-time PCR was performed to measure the mRNA copy number of EGFP, as well as of three host cell genes (FGFR, HSPG, and integrin) that are required for viral entry. As shown in Table 1, addition of DXR did not affect GFP DNA copy number but significantly increased EGFP mRNA levels in a time-dependent manner. In contrast, mRNA levels of FGFR, HSPG, and integrin remained unaltered at most time points, indicating that viral entry is not significantly enhanced by the addition of chemotherapeutic drugs during infection. These results indicated that the increase of rAAV2-delivered GFP expression seemed to be mediated by upregulation on a transcriptional level. 
Table 1.
 
DNA Copy and mRNA Expression in Cells Infected with rAAV2-CNTF plus DXR
Table 1.
 
DNA Copy and mRNA Expression in Cells Infected with rAAV2-CNTF plus DXR
In Vivo Verification of DXR-Enhanced rAAV2 Transduction and Transgene Expression
As the in vitro data were encouraging, testing was performed to determine if the results could be reproduced in vivo in normal SD rats. Recombinant AAV2-EGFP was injected into the subretinal space of normal SD rats, with or without coadministration of DXR (1 μg in 1 μL PBS). GFP expression was evaluated under a fluorescent stereoscope at 1, 7, 60, and 240 days postinjection. In rAAV2-EGFP–only injected retinas, weak GFP fluorescence was detectable 7 days postinfection, peaking at 60 days and declining at 240 days. In contrast, the combination of rAAV2-EGFP and DXR significantly increased both the onset and the absolute level of GFP expression. As shown in Figure 5A, strong GFP fluorescence was readily detectable 1 day postinjection, peaked at 60 days, and persisted up to 240 days. The frozen sections of retinas that received rAAV2-EGFP alone or rAAV2-EGFP plus DXR showed a similar GFP expression pattern except for differential brightness. All retinal cells in the injected area, including RPE, photoreceptor, inner nuclear layer, and ganglion cells, expressed GFP (data not shown). These data indicated that DXR enhanced both the level and the duration of rAAV2-mediated transgene expression. To verify these results with a different reporter gene, subretinal injection of rAAV2-Luc into the right eye and rAAV2-Luc in combination with DXR into the left eye of Balb/c mice (n = 5) was performed, and luciferase activity was assessed using bioluminescent in vivo imaging. In concordance with the previous data, the left eyes of Balb/c mice that had been coinjected with rAAV2-Luc and DXR exhibited significant enhancement, acceleration, and longer duration of luciferase activity compared to the right eyes injected with rAAV2-Luc only (Figs. 5B, 5C). 
Figure 5.
 
DXR enhances rAAV2-mediated retinal transgene expression in vivo. (A) Representative GFP expression in the fundus of SD rats after subretinal injection of rAAV2-GFP (left eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL PBS) and coinjection of rAAV2-GFP and DXR (right eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL DXR [1 μg/μL]) at indicated time points. (B) Representative bioluminescence images taken at indicated time points of Balb/c mice after subretinal coinjection of rAAV2-Luc and DXR (left eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL DXR [1 μg/μL]) or rAAV2-Luc only (right eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL PBS). (C) Time course of Luc expression in vivo after coinjection of rAAV2-Luc and DXR versus rAAV2-Luc only. N = 5.
Figure 5.
 
DXR enhances rAAV2-mediated retinal transgene expression in vivo. (A) Representative GFP expression in the fundus of SD rats after subretinal injection of rAAV2-GFP (left eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL PBS) and coinjection of rAAV2-GFP and DXR (right eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL DXR [1 μg/μL]) at indicated time points. (B) Representative bioluminescence images taken at indicated time points of Balb/c mice after subretinal coinjection of rAAV2-Luc and DXR (left eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL DXR [1 μg/μL]) or rAAV2-Luc only (right eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL PBS). (C) Time course of Luc expression in vivo after coinjection of rAAV2-Luc and DXR versus rAAV2-Luc only. N = 5.
Chemotherapeutic Drugs Can Enhance the Protective Effect of an rAAV2-Mediated Neuronal Survival Factor in Rat Photoreceptor Cells
CNTF is a survival factor for neuronal cells and has been shown to protect photoreceptor cells when coexpressed or upregulated. 9,10 APRE-19 cells were infected with rAAV2-CNTF with or without addition of DXR, VP-16, or Ara-C, and expression levels were determined using Western blot analysis. As a positive control, cell extracts from cells coinfected with the helper virus Ad5, which can promote enhanced rAAV2 expression, were used. 33 As can be seen in Figure 6A, addition of DXR, VP-16, or Ara C significantly increased CNTF expression in APRE-19 cells. 
Figure 6.
 
Enhanced rAAV2-delivered CNTF expression resulting in increased thickness and cell density of the ONL. CNTF expression and photoreceptor survival were analyzed in RCS rats 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) alone or combined with chemotherapeutic drugs. (A) Left panel: Western blot analysis for CNTF expression of cell extracts from RCS rat retinas collected 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) in the presence of chemotherapeutic drugs as indicated. β-Actin was used as a loading control. Right panel: Corresponding densitometric Western blot analysis of CNTF protein levels. (B) Left panel: Representative images of retina sections after H&E staining. RCS rats were sacrificed 8 weeks after subretinal injection of PBS or rAAV2-CNTF (1. 9 vg/eye) with or without DXR (1 μL DXR [1 μg/μL]), and retinas were collected and stained with H&E. Right panel: Corresponding manual cell count of the outer nuclear layer (ONL) expressed as nuclei per micrometer. N = 5 for each group.
Figure 6.
 
Enhanced rAAV2-delivered CNTF expression resulting in increased thickness and cell density of the ONL. CNTF expression and photoreceptor survival were analyzed in RCS rats 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) alone or combined with chemotherapeutic drugs. (A) Left panel: Western blot analysis for CNTF expression of cell extracts from RCS rat retinas collected 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) in the presence of chemotherapeutic drugs as indicated. β-Actin was used as a loading control. Right panel: Corresponding densitometric Western blot analysis of CNTF protein levels. (B) Left panel: Representative images of retina sections after H&E staining. RCS rats were sacrificed 8 weeks after subretinal injection of PBS or rAAV2-CNTF (1. 9 vg/eye) with or without DXR (1 μL DXR [1 μg/μL]), and retinas were collected and stained with H&E. Right panel: Corresponding manual cell count of the outer nuclear layer (ONL) expressed as nuclei per micrometer. N = 5 for each group.
To test whether coadministration of chemotherapeutic drugs during infection with rAAV2-CNTF could enhance its protective effect in vivo, the retinas of RCS rats, which serve as an animal model for inherited degenerative retinopathy, were injected with either rAAV2-CNTF alone or in combination with DXR (0.1 μg/mL). After sacrifice of the animals, the retinas were histologically analyzed for thickness of the photoreceptor layer and relative number of nuclei in the ONL as a surrogate for the protective effect of the treatment. Recombinant AAV2-CNTF injection alone had only a slight effect on the thickness of the photoreceptor layer compared to noninjected or PBS-injected control retinas, whereas combination treatment with DXR increased the overall thickness (2-fold) (Fig. 6B, left panel). In addition, the number of nuclei per micrometer of the ONL was significantly higher in retinas that had been injected with the rAAV2-CNTF + DXR (χ = 8.05 ± 0.28) than in retinas injected with PBS (χ = 1.40 ± 0.23), injected with rAAV2-CNTF alone (χ = 3.96 ± 0.16), or noninjected (χ = 1.36 ± 0.21) (P < 0.05 for paired comparisons, Fig. 6B, right panel). 
Discussion
This study tested whether coadministration of chemotherapeutic agents such as DXR, Ara-C, VP-16, and DDP during infection with rAAV2 could improve rAAV2-mediated transgene expression. Such an improvement in expression levels is of significance since low expression of virally delivered genes presents one of the biggest challenges in modern gene therapy. The study showed that addition of chemotherapeutic agents to the infection mixture markedly increased rAAV2-mediated transgene expression in cultured hRPE cells as well as in vivo. 
Several groups have tried to improve the limited transgene expression of rAAV2 vectors in gene therapy, and positive results have been reported for several chemicals such as hydroxyurea and DNA-damaging treatments including γ and UV irradiation. 2325 However, thus far the severe cytotoxic nature of these treatments presents a major concern and prevents them from being used in the clinic. The present study tested cytotoxic chemotherapeutic drugs that are widely used in anticancer treatment, however at doses well below those used in the clinical setting. Interestingly, the most pronounced enhancement of transduction efficacy was achieved at doses at which there was no measurable cytotoxic effect. Further dose escalation did not result in better expression levels in vitro. However, for in vivo applications, further dose finding studies are required to optimize efficacy and at the same time limit toxicity to a minimum. 
It was hypothesized that the gentle cell cycle delay induced by these drugs at moderate doses might provide the virus ample time for the conversion from single- to double-stranded DNA, which is considered the rate-limiting step in AAV-mediated transduction. Interestingly, the timing of drug application seemed to be of relevance, since drug treatment was most effective when applied prior to infection. In contrast, the specific mode of action of the chemotherapeutic drugs did not seem to be important: DXR is a DNA intercalating agent; Ara-C interferes with DNA synthesis; VP-16 causes double-strand breaks; and DPP introduces DNA crosslinks. However, all of these drugs are known to cause a cell cycle arrest or delay. 
Since chemotherapeutic drugs are known to induce intrinsic cellular DNA repair systems, it can be speculated that active DNA repair mechanisms might contribute to the dramatic enhancement of the expression of rAAV2-carried genes in retinal cells. Interestingly, DNA copy number of rAAV2, as well as mRNA levels of receptors required for viral entry receptor upregulation, was not affected by the drug treatment, further indicating that viral entry that translates to infection efficiency per se is not facilitated by addition of chemotherapeutic drugs. Rather, the enhancement effect seems to occur in the target cell after infection has successfully taken place. The observation that by adding chemotherapeutic drugs not only the GFP intensity but also the percentage of positive cells increased can be explained with the assumption that although the total number of positively infected cells remains unaltered, the percentage of cells at which GFP expression remains at undetectable levels is smaller. These results are consistent with previous findings showing that addition of HDAC inhibitors could enhance the expression of rAAV-carried genes at the mRNA level. 24 However, the exact underlying molecular mechanisms remain to be determined. 
Previous studies have shown that both adenovirus- and rAAV2-delivered CNTF can protect photoreceptor cells and that the effect of adenovirus-delivered CNTF is superior to that with injection of recombinant CNTF alone. 9,10 However, adenoviruses are prone to induce tissue immune responses, and Ad-mediated transgene expression is of shorter duration. 9  
The present study made it possible demonstrate that intraocular injection of rAAV2-CNTF in the presence of chemotherapeutics drugs remarkably increased thickness and cell density of the ONL in rats suffering from inherited retinal degeneration. These data indicate that this strategy can be therapeutically exploited. However, an ideal surrogate for the therapeutic effect would be an assessment of eye vision, which is difficult in rat animal models, but would be very easy if applied to human patients. 
In conclusion, the results of this study provide the first evidence that low concentrations of chemotherapeutic drugs coadministered during infection with rAAV2 dramatically increase and prolong the expression of rAAV2-delivered transgenes in vitro as well as in vivo. Subretinal injection of CNTF using the strategy described here resulted in a pronounced protection effect without detectable toxicity, indicating that the strategy can be therapeutically exploited. The authors believe that this strategy provides a simple and feasible method to improve retinal gene therapy and could be extended to target the endothelium of the retinal blood vessels and inner retinal cells, providing an optimistic perspective to the use of retinal gene therapy in the clinic and warranting further preclinical studies. 
Acknowledgments
The authors thank Xiafang Chen, Kuangcheng Xie, and the entire staff at the experimental center of First People's Hospital for their technical assistance. 
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Footnotes
 Supported by grants from National Basic Research Program of China (2010CB529902), Science and Technology Committee of Shanghai (064119539), Leading Medical Talent of Shanghai (040308), and National Natural Science Foundation for Outstanding Youth (30325043, 30428015).
Footnotes
5  These authors contributed equally to this work.
Footnotes
 Disclosure: S. Zhang, None; J. Wu, None; X. Wu, None; P. Xu, None; Y. Tian, None; M. Yi, None; X. Liu, None; X. Dong, None; F. Wolf, None; C. Li, None; Q. Huang, None
Figure 1.
 
Chemotherapeutic drugs increase rAAV2-delivered GFP expression in human retinal pigmented epithelium cells. (A) Representative fluorescence (upper panel) and bright field (lower panel) images of ARPE-19 cells after infection with rAAV2-EGFP alone (a, b), or coadministration of 0.1 μg/mL DXR (c, d), 0.14 μg/mL Ara-C (e, f), 1 μg/mL VP-16 (g, h), or 20 μg/mL DDP (i, j). Imaging was performed 7 days postinfection. (B) FACS analysis of ARPE-19 cells 7 days after infection with rAAV2-EGFP at a viral titer of either 1. 2 , 1. 3 , or 1. 4 vg/cell with or without the presence of chemotherapeutic drugs as indicated. Percentage of GFP-positive cells (B) and mean intensity of GFP fluorescence (C) are plotted. Data are presented as mean ± SD. N ≥ 3. BF, bright field.
Figure 1.
 
Chemotherapeutic drugs increase rAAV2-delivered GFP expression in human retinal pigmented epithelium cells. (A) Representative fluorescence (upper panel) and bright field (lower panel) images of ARPE-19 cells after infection with rAAV2-EGFP alone (a, b), or coadministration of 0.1 μg/mL DXR (c, d), 0.14 μg/mL Ara-C (e, f), 1 μg/mL VP-16 (g, h), or 20 μg/mL DDP (i, j). Imaging was performed 7 days postinfection. (B) FACS analysis of ARPE-19 cells 7 days after infection with rAAV2-EGFP at a viral titer of either 1. 2 , 1. 3 , or 1. 4 vg/cell with or without the presence of chemotherapeutic drugs as indicated. Percentage of GFP-positive cells (B) and mean intensity of GFP fluorescence (C) are plotted. Data are presented as mean ± SD. N ≥ 3. BF, bright field.
Figure 2.
 
Optimization of doxorubicin dosage. (A) Representative fluorescence images of ARPE-19 cells 7 days after infection with rAAV2-EGFP in the presence of increasing amounts of DXR as indicated. (B, C) FACS analysis of APRE-19 cells at different time points after infection with rAAV2-EGFP (1. 4 vg/cell). The percentage of GFP-positive cells (B) as well as the mean overall GFP intensity (C) is shown for different concentrations of DXR present at the time of infection. Data are presented as mean ± SD. N ≥ 3.
Figure 2.
 
Optimization of doxorubicin dosage. (A) Representative fluorescence images of ARPE-19 cells 7 days after infection with rAAV2-EGFP in the presence of increasing amounts of DXR as indicated. (B, C) FACS analysis of APRE-19 cells at different time points after infection with rAAV2-EGFP (1. 4 vg/cell). The percentage of GFP-positive cells (B) as well as the mean overall GFP intensity (C) is shown for different concentrations of DXR present at the time of infection. Data are presented as mean ± SD. N ≥ 3.
Figure 3.
 
Cytarabine (Ara-C) and etoposide (VP-16) increase AAV2-mediated transgene expression. (A) FACS analysis showing the percentage of GFP-positive cells and the mean GFP fluorescence intensity in APRE-19 cells that had been infected with rAAV2-EGFP (1. 4 vg/cell) in the presence of different concentrations of VP-16 (upper panel) and Ara-C (lower panel) at various time points. Data are presented as mean ± SD. N ≥ 3. (B) Luciferase activity (RL) of RPE cells infected with rAAV2-Luc alone or in the presence of different concentrations of VP-16. Luciferase activity was measured 3 days after infection. Data are presented as mean ± SD. N ≥ 3. (C) Representative Western blot of GFP expression in ARPE-19 cells 7 days after infection with rAAV2-EGFP (1. 4 vg/cell) with or without the presence of different concentrations of Ara-C. β-Actin was used as loading control.
Figure 3.
 
Cytarabine (Ara-C) and etoposide (VP-16) increase AAV2-mediated transgene expression. (A) FACS analysis showing the percentage of GFP-positive cells and the mean GFP fluorescence intensity in APRE-19 cells that had been infected with rAAV2-EGFP (1. 4 vg/cell) in the presence of different concentrations of VP-16 (upper panel) and Ara-C (lower panel) at various time points. Data are presented as mean ± SD. N ≥ 3. (B) Luciferase activity (RL) of RPE cells infected with rAAV2-Luc alone or in the presence of different concentrations of VP-16. Luciferase activity was measured 3 days after infection. Data are presented as mean ± SD. N ≥ 3. (C) Representative Western blot of GFP expression in ARPE-19 cells 7 days after infection with rAAV2-EGFP (1. 4 vg/cell) with or without the presence of different concentrations of Ara-C. β-Actin was used as loading control.
Figure 4.
 
Recombinant AAV2-mediated transgene expression is affected by the relative timing of drug administration. FACS analysis of APRE-19 cells at various time points after infection with rAAV2-EGFP and DXR 0.1, μg/mL, administered either 24 hours prior to, simultaneously with, or 24 hours after infection. Shown are the percentage of GF-positive cells (A) and the mean overall GFP fluorescence intensity (B) at different time points after infection as indicated.
Figure 4.
 
Recombinant AAV2-mediated transgene expression is affected by the relative timing of drug administration. FACS analysis of APRE-19 cells at various time points after infection with rAAV2-EGFP and DXR 0.1, μg/mL, administered either 24 hours prior to, simultaneously with, or 24 hours after infection. Shown are the percentage of GF-positive cells (A) and the mean overall GFP fluorescence intensity (B) at different time points after infection as indicated.
Figure 5.
 
DXR enhances rAAV2-mediated retinal transgene expression in vivo. (A) Representative GFP expression in the fundus of SD rats after subretinal injection of rAAV2-GFP (left eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL PBS) and coinjection of rAAV2-GFP and DXR (right eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL DXR [1 μg/μL]) at indicated time points. (B) Representative bioluminescence images taken at indicated time points of Balb/c mice after subretinal coinjection of rAAV2-Luc and DXR (left eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL DXR [1 μg/μL]) or rAAV2-Luc only (right eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL PBS). (C) Time course of Luc expression in vivo after coinjection of rAAV2-Luc and DXR versus rAAV2-Luc only. N = 5.
Figure 5.
 
DXR enhances rAAV2-mediated retinal transgene expression in vivo. (A) Representative GFP expression in the fundus of SD rats after subretinal injection of rAAV2-GFP (left eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL PBS) and coinjection of rAAV2-GFP and DXR (right eye, 1 μL rAAV2-EGFP [1 × 1. 9 vg] plus 1 μL DXR [1 μg/μL]) at indicated time points. (B) Representative bioluminescence images taken at indicated time points of Balb/c mice after subretinal coinjection of rAAV2-Luc and DXR (left eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL DXR [1 μg/μL]) or rAAV2-Luc only (right eye, 0.5 μL rAAV2-Luc [5 × 1. 8 vg] plus 0.5 μL PBS). (C) Time course of Luc expression in vivo after coinjection of rAAV2-Luc and DXR versus rAAV2-Luc only. N = 5.
Figure 6.
 
Enhanced rAAV2-delivered CNTF expression resulting in increased thickness and cell density of the ONL. CNTF expression and photoreceptor survival were analyzed in RCS rats 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) alone or combined with chemotherapeutic drugs. (A) Left panel: Western blot analysis for CNTF expression of cell extracts from RCS rat retinas collected 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) in the presence of chemotherapeutic drugs as indicated. β-Actin was used as a loading control. Right panel: Corresponding densitometric Western blot analysis of CNTF protein levels. (B) Left panel: Representative images of retina sections after H&E staining. RCS rats were sacrificed 8 weeks after subretinal injection of PBS or rAAV2-CNTF (1. 9 vg/eye) with or without DXR (1 μL DXR [1 μg/μL]), and retinas were collected and stained with H&E. Right panel: Corresponding manual cell count of the outer nuclear layer (ONL) expressed as nuclei per micrometer. N = 5 for each group.
Figure 6.
 
Enhanced rAAV2-delivered CNTF expression resulting in increased thickness and cell density of the ONL. CNTF expression and photoreceptor survival were analyzed in RCS rats 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) alone or combined with chemotherapeutic drugs. (A) Left panel: Western blot analysis for CNTF expression of cell extracts from RCS rat retinas collected 8 weeks after subretinal injection of rAAV2-CNTF (1. 9 vg/eye) in the presence of chemotherapeutic drugs as indicated. β-Actin was used as a loading control. Right panel: Corresponding densitometric Western blot analysis of CNTF protein levels. (B) Left panel: Representative images of retina sections after H&E staining. RCS rats were sacrificed 8 weeks after subretinal injection of PBS or rAAV2-CNTF (1. 9 vg/eye) with or without DXR (1 μL DXR [1 μg/μL]), and retinas were collected and stained with H&E. Right panel: Corresponding manual cell count of the outer nuclear layer (ONL) expressed as nuclei per micrometer. N = 5 for each group.
Table 1.
 
DNA Copy and mRNA Expression in Cells Infected with rAAV2-CNTF plus DXR
Table 1.
 
DNA Copy and mRNA Expression in Cells Infected with rAAV2-CNTF plus DXR
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