Construction and identification of recombinant adeno- associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein*☆

Huang Xiang-hui1, Shi Zhi-bin1, Wang Kun-zheng1, Dang Xiao-qian1, Yang Pei1, Yu Peng-bo2

1Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China; 2Laboratory of Virus, Shanxi Provincial Center for Disease Control and Prevention, Xi’an   710054, Shanxi Province, China

Huang Xiang-hui☆, Studying for doctorate, Attending Physician, Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China
drhxh@163.com

Correspondence to: Shi Zhi-bin, Doctor, Lecturer, Attending Physician, Department of Orthopaedics, Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an   710004, Shaanxi Province, China
jackky9999@ sohu.com

Supported by: the National Natural Science Foundation of China, No. 30600624*

Received:2008-01-08
Accepted:2008-03-11
(08-50-1-185/ZS)

Huang XH, Shi ZB, Wang KZ, Dang XQ, Yang P, Yu PB. Construction and identification of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):
5755-5758(China)
[www.zglckf.com/ zglckf/ ejournal/ upfiles/08-29/ 29k-5755(ps).pdf]

Abstract
BACKGROUND: Vascular endothelial growth factor (VEGF) can specifically promote the division and proliferation of endothelial cells and the revascularization, finally induce angiopoiesis. Recently, VEGF-based gene therapy has been gradually used in clinical trials, but some limits on usually used vectors deserve further studies, including the low transfection efficiency of plasmid vector, the immunogenicity of adnovirus vector to host cells and the potential risk of infection.
OBJECTIVE: To construct the non-pathogenic recombinant adeno-associated virus (AAV) co-expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label, and measure the virus titer and assess its biological activity.
DESIGN, TIME AND SETTING: The open experiment was performed at the Virus Laboratory of Shanxi Provincial Center for Disease Control and Prevention from March to September 2007.
MATERIALS: AAV-293 virus packaging cell line, AAV HT-1080 cells were purchased from Stratagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. AAV Helper Free System (pAAV-IRES-GFP vector containing GFP label) was purchased from Stratagene, USA. Plasmid pUC18-hVEGF165 was constructed previously by Dr.Shi from Department of Orthopaedics of Second Affiliated Hospital of Xi’an Jiaotong University.
METHODS: The hVEGF165 gene from plasmid pUC18-hVEGF165 was amplified and inserted into plasmid pAAV-IRES-hrGFP. Then recombinant plasmid pAAV-hVEGF165-IRES-hrGFP, pAAV-RC and pAAV-Helper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells and further concentrated and purified. The recombinant virus infected the AAV-HT1080 cells, the virus titer was measured by fluorescence counting, and the recombinant AAV-hVEGF165-IRES-hrGFP was verified by the amplification of the exogenous gene of hVEGF165 from virus genome.
MAIN OUTCOME MEASURES: Virus packaging efficiency was monitored under the fluorescence microscope. Virus titer was measured by fluorescent counting. The packaging of recombinant virus was determined by the amplification of the exogenous hVEGF165 gene.
RESULTS: The amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-hrGFP was confirmed by double digestion. The system provided a high packaging ratio of over 95% and the purified recombinant virus had a high titer of 5.5×1011 vp/mL. The recombinant virus was confirmed by exogenous human VEGF165 gene from virus genome.
CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP simultaneously carrying hVEGF165 and GFP label is successfully constructed, with a high titer and satisfying biological activity.

INTRODUCTION

Presently, VEGF-based gene therapy has been gradually used in clinical trial to treat disease of orthopedics such as osteonecrosis and osteoanagenesis defects, but there are some limits on usually used vectors, including the low transfection efficiency, immunogenicity to host cells and the potential hazards of infection. Adeno-associated virus (AAV) has many natural features that make it attractive for a human viral vector. In this study, we developed a recombinant AAV vector system capable of co-expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label, which may offer the fundament for in vitro and in vivo experiments of hVEGF165 expression and provide a new method for gene therapy of bone regeneration.

MATERIALS　AND　METHODS

Materials
This study was conducted at the Virus Laboratory of Shanxi Provincial Center for Disease Control and Prevention between March 2007 and September 2007. AAV Helper Free System (pAAV-MCS vector, pAAV-IRES-GFP vector, pAAV-RC plasmid, and pHelper plasmid) and AAV-293 packaging cell line, AAV HT-1080 cells were purchased from Stratagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. Plasmid pUC18-hHVEGF165 was constructed previously by Dr. Shi. Restriction enzyme and DNA marker were purchased from TAKARA, China. The primers were synthesized by Augtc, China. Plasmid Mini preps and Agarose Gel DNA Extraction Kit were purchased from Qiagen, Germany. DMEM-H Growth Medium and fetal bovine serum were purchased from GIBIC, USA. Calcium Phosphate Cell Transfection Kit was purchased from Invitrogen.

Methods
Construction of the recombinant expression plasmid pAAV-hVEGF165-IRES-GFP
Primers were designed to amplify human VEGF165 gene according to the sequence published at PUBMED

(NM_003376). Cleavage site of EcoR I and XhoI was respectively added to the forward and reverse primer. Primer F: 5'-CCGAATTCATGAACTTTCTGCTGTCTTG-3'，primer R: 5'-GGCCTCGAGTCACCGCCTCGGCTTGTC-3'. The pUC18-hVEGF165 was used as a template to conduct polymerase chain reaction (PCR). The PCR products were identified and extracted by 1% agarose gel electrophoresis. The purified PCR products were directly subcloned into the multiple cloning sites of pAAV-IRES-GFP, which was also digested with ECOR I and XhoI. After verifying with cleavage and PCR screening, the positive recombinant clone was named pAAV-hVEGF165-IRES-GFP.

Packaging of recombinant AAV co-expressing hVEGF165 and GFP
AAV-293 cells were cultured in high glucose DMEM supplemented with 10 % fetal bovine serum at 37 ℃ and 5 % CO2. Plate the cells at 100 mm tissue culture plate 48 hours prior to transfection. With calcium phosphate transfection protocol, the triple transfection of pAAV-hVEGF165-IRES-GFP, pAAV-RC and pHelper was performed to package the recombinant AAV-hVEGF165-IRES-GFP (experimental group); the triple transfection of plasmid pAAV-IRES-hrGFP, pAAV-RC and pHelper was used to construct rAAV-IRES-GFP, which was labeled with GFP and served as viral production parallel group; and a negative control group was performed for simultaneous observation by substituting the recombinant AAV expression plasmid with 10 μL TE buffer. The progress of AAV particle production was monitored under inverted microscope by observing phenotypic changes and cell-cytotoxic reaction of the AAV-293 cells, and the expression of GFP in AAV-293 was detected under the fluorescence microscope at 24, 48 and 72 hours after transfection. The ratio of cells labeled with GFP was calculated to ascertain the packaging efficiency. After 72 hours incubation, the AAV-293 cells and culture medium was collected together to get viral particles. The cell suspension was subject to four rounds of freeze/thaw by alternating the tubes between dry ice-ethanol bath and 37 ℃ water bath. After centrifuged at 10 000 g for 10 minutes, the supernatant containing primary virus stock was collected. The virus stock was further concentrated and purified with chloroform/ PEG8000/ chloroform protocols [1].

Viral titer measurement of recombinant AAV
AAV-HT1080 cells were cultured in high glucose DMEM as routine method for viral titer measurement. The recombinant viral stock was diluted with DMEM over a 10 fold dilution series, and each dilution was added to infect AAV-HT1080 cells cultured in 24-well plates. Each dilution was performed in triplicate and a virus-free well was performed as a negative control. The infection was incubated at 37 ℃ for 2 hours, during which the plates were swirled gently at 30-minute intervals. Forty-eight hours after infection, GFP expression in AAV-HT1080 was detected under the fluorescence microscope and fluorescing cells of each well was counted. Subsequently, the well of which the number of fluorescing cells between 10 and 100 was selected to calculate the viral titer (vp/mL=n×dilution multiple).
Determination of the recombinant virus rAAV-hVEGF165-IRES-GFP
To confirm that hVEGF165 gene was successfully inserted into the recombinant AAV, we verified the recombinant virus by PCR of the exogenous interest genes. The virus rAAV-hVEGF165-IRES-GFP and rAAV-IRES-GFP were deliquated, DNase and RNase were added with a final concentration of 1 mg/L and incubated for 30 minutes to digest the residual DNA and RNA. Subsequently, protease K was added to split outer shell of the AAV and the virus genome was extracted using QIAamp VIRUS MINI KIT. 1μL of virus genome was used as a template to conduct PCR with hVEGF165 primer. The PCR products were identified by 1% agarose gel electrophoresis.

RESULTS

Construction of the recombinant expression plasmid pAAV-hVEGF165-IRES-GFP
The hVEGF165 gene was successfully amplified and verified from plasmid PUC18-VEGF165. The amplified hVEGF165 gene was successfully inserted into MCS of pAAV-IRES-GFP and the recombinant bicistronic expression plasmid pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The size of each fragment was exactly in accordance with expectation (Figure 1).

Packaging efficiency of recombinant AAV
In the experimental group and parallel group, the GFP expression in AAV-293 was detected 24 hours after transfection, and the proportion of cells expressing GFP increased rapidly as time goes by. The intension of fluorescence also strengthened gradually. The ratio of cell labeled with GFP reached a peak of 95% at 72 hours after transfection. There were no phenotypic changes and cell-cytotoxic reaction in the negative control group, and GFP expression in AAV-293 was negative.

Viral titer measurement of recombinant AAV
The GFP expression in AAV-HT1080 was detected under the fluorescence microscope 48 hours after AAV infection, and the number of fluorescing cells of each well was correlated with the concentration of virus administered. The efficiency of infection was 90%. The viral titer was calculated as 5.5×1011 vg/mL.

Verify of rAAV-hVEGF165-IRES-GFP by PCR of the exogenous interest gene
The 600 bp target band was separated by 1% agarose gel electrophoresis in the experimental group, which was exactly in accordance with the length of exogenous hVEGF165 gene fragment. There was no target band in the rAAV-IRES-GFP parallel group (Figure 2).

DISCUSSION

Bone renovation is a complicated process involved with many kinds of cytokines, of which VEGFs have important roles and are surveyed extensively. VEGF is one of the most important cytokines of angiogenesis. VEGF could specifically promote the division and growth of the vascular endothelial cells and finally induce angiopoiesis[2]. VEGF was essential for bone formation and reparation as the whole process of osteogenesis was closely associated with VEGF-mediated angiogenesis[3]. Endothelial cells could be stimulated by VEGF and release cytokines IGF-1, ET-1, which cohered to the corresponding receptor located on the membrane of osteoblasts and accelerated bone formation in result[4]. Moreover, endogenous or exogenously added VEGF directly attracted endothelial cells and osteoclasts and enhanced the differentiation of osteoblasts by activating VEGFR-3 expressed in osteoblasts, resulting in regulating bone formation and remodeling[5-7]. VEGF165 had chemotactic effects on both human osteoblastic and endothelial cells, indicating that it was directly involved in promoting angiogenesis and osteogenesis by recruiting osteoblasts and enclothelial cells via chemotaxis[8]. Exogenous VEGF could enhance blood vessel formation, ossification, and new bone maturation in mouse femur fractures, and promote bony bridging of a rabbit radius segmental gap defect; while treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures [9]. On the other hand, osteoblasts can be induced by most osteoinductive growth factors such as bone morphogenetic proteins (BMPs) to express VEGF. BMPs have definite effects of regulating chondrogenesis and skeletogenesis and they are the only signaling molecules, which can singly induce bone formation at orthotopic and heterotopic sites. Presently, extensive studies demonstrated that BMPs also stimulated osteoblastogenesis and angiogenesis through the production of VEGF-A[10-11]. In the presence of a VEGF-A antibody, both non-stimulated and BMP- stimulated angiogenesis were arrested [12]. Inhibition of VEGF blocked BMP2 induced angiogenesis, BMP7 induced primary osteoblast differentiation, and BMP4 induced bone formation[13-15]. These results strongly suggest that angiogenesis and bone induction is a correlated and coordinated process during bone formation and their effects may be enhanced by each other. VEGF participated in the BMP induced osteogenesis by taking part in bone cell differentiation and by promoting angiogenesis at the site of bone formation, and BMP regulated osteogenesis and bone renovation by increasing VEGF expression of osteoblast[16]. Transforming growth factor (TGF)-beta 1 also induced a dose-dependent VEGF expression in osteoblasts and osteoblast-like cells and they both followed similar patterns of mRNA and protein expression during rat mandibular fracture healing[17]. In summary, bone formation is a complicated and coordinated process in which VEGFs involved and may be essential, and up-regulation of the VEGF expression may greatly enhance the process of bone formation and reparation.
With the rapid development of gene therapy, the method of introduction of VEGF gene into cells to treat disease had been gradually used in clinical trials[18-19]. However, there are some limits on usually used vectors, including the low transfection efficiency, immunogenicity to host cells and the potential hazards of infection. Taken together, choosing a safe and effective vector system to transfer and correctly express target gene is an important issue in gene therapy. AAV has many natural features that make it attractive for a human viral vector. All these features make AAV attractive and efficient for gene transfer in vitro and local injection in vivo. Using the AAV helper-free system in combination with IRES sequence, we successfully constructed AAV co-expressing hVEGF165 and GFP label. The viral titer was measured by infecting AAV-HT1080 optimized by Stratagene, which precluded the disturbance due to empty viral particle and difference of cell liability. The recombinant AAV has a high titer of 5.5×1011 vp/mL, meeting the requirements of experiments in vitro and in vivo. GFP fluorescence in the system may be used to monitor the process of AAV production and measure the titer of the recombinant virus directly. In addition, since both proteins are translated from the same transcript, GFP expression may be used to ascertain the infection efficiency for the desired target cells and also serves as a useful expression marker for the inserted gene of interest. The construction of rAAV-hVEGF165-IRES-GFP lays a basement for in vitro and in vivo experiments of hVEGF165 expression and may provide a new method for bone regeneration therapeutics.

REFERENCES

1 Xin ZC, Kim EK, Tian ZJ, et al. Icariin on relaxation of corpus cavernosm smooth muscle. Chin Sci Bull 2001;46:485-489
2 Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton.J Bone Miner Res 2006;21(2):183-192
3 Carano RAD, Filvaroff EH. Angiogenesis and bone repair. Drug Discovery Today 2003;8(21):980-989
4 Mayer H, Bertram H, Lindenmaier W, et al. Vascular endothelial growth factor(VEGF-A)expression in human mesenchyreal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation. J Cell Biochem 2005;95(4):827-839
5 Deckers MM, Karperien M, Vander B, et al. Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology 2000;141(5): 1667-1674
6 Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 2007;86(10):937-950
7 Orlandini M, Spreafico A, Bardelli M, et al. Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation. J Biol Chem 2006;281(26):17961-17967
8 Li G, Cui Y, McIlmurray L, et al. rhBMP-2, rhVEGF(165), rhPTN and thrombin-related peptide, TP508 induce chemotaxis of human osteoblasts and microvascular endothelial cells. J Orthop Res 2005;23 (3):680-685
9 Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Nat Acad Sci 2002;99 (15):9656-9661
10 FU DH, Yang SH, Yang C,et al. Dose-dependent effect of rhBMP-2 on VEGF expression in fetal mouse osteoblasts. Journal of Traumatic Surgery 2006;8(6):541-545
11 FU XH, Yang SH, Yang C, et al. Time-dependent Effect of rhBMP-2 Promoting the VEGF Expression in Fetal Mouse Osteoblasts. Chinese Journal of Bone and Joint Injury 2006;21(8):628-630
12 Deckers MM, Vanbezooijen RL, Vanderhorgst G, et al. Bone morphogenetic proteins stimulate angiogenesis through osteoblast- derived vascular endothelial growth factor A. Endocrinology 2002;143 (4): 1545-1553
13 Yeh LC, Lee JC. Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. Molecular and Cellular Endocrinology 1999;153(1-2): 113-124
14 Claffey KP, Abrams K, Shih SC, et al. Fibroblast growth factor 2 activation of stromal cell vascular endothelial growth factor expression and angiogenesis. Lab Invest 2001; 81(1):61-75
15 Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest 2002;110 (4):751-759
16 Zhou ZL, Wang CL, Zhao Y, et al. Synergistic effect of bone morphogenetic protein and vascular endothelial growth factor in bone tissue engineering. Journal of Clinical Rehabilitative Tissue Engineering Research 2007;11(36):7227-7230
17 Saadeh PB, Mjehrara BJ, Steinbrech DS, et al. Transforming growth factor-beta1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am J Physil 1999;27(6):628-637
18 Yla-Herttuala S. An update on angiogenic gene therapy: vascular endothelial growth factor and other directions. Curr Opin Mol Ther 2006;8(4):295-300
19 Polak J, Hench L. Gene therapy progress and prospects: in tissue engineering. Gene Ther 2005;12(24):1725-1733