四种病毒载体对人成纤维细胞转染效率的研究
摘要
基因治疗是指将目的基因用基因转移技术导入目的细胞进行表达,使其获得特定的功能,从而达到治疗的目的[1]。基因治疗由三个方面组成:受体细胞、载体以及目的基因。
皮肤成纤维细胞(dermal fibroblasts,DFs)获取方便,可以在体外大量扩增,容易进行遗传改造,并且具有容易移植和不易转化的优点,因此DFs成为目前基因治疗应用较为广泛的靶细胞,本论文亦将此细胞作为研究对象。
目前,基因治疗面临的瓶颈问题在于转染效率低下,而决定基因治疗转染效率的关键点之一就在于载体的选择。目前基因治疗所应用的载体主要包括病毒载体和非病毒载体,其中应用最多的是病毒载体,包括逆转录病毒,腺病毒,腺相关病毒,疱疹病毒和慢病毒[2],其中前四种已经被美国FDA批准应用于临床。在人类基因治疗常用的病毒载体中,腺相关病毒( Adeno-associated viruses, AAV)的安全优势以及长效表达的优点[3-4]使其成为了基因稳定转染的宠儿。而腺病毒(Adenovirus, Ad)载体则因具有感染效率和外源基因表达水平高、高滴度制备较简单等特点而被广泛用于基因治疗研究和临床试验中[5-6]。因此,本论文选择AAV和Ad为研究对象。由于病毒载体的衣壳蛋白是决定其对靶细胞嗜性的主要因素,进行衣壳蛋白改造后的载体有望改变载体亲嗜性而提高转染效率。我们在选用常规使用的AAV2和Ad5的基础上,还选用了将AAV2的ITRs包被进入AAV1的衣壳蛋白形成的嵌合型载体——AAV2/1,以及将Ad35载体的纤突整合到Ad5载体上形成的Ad5/F35作为研究对象,通过比较他们的转染效率来筛选适合DFs的载体。
本学位论文以eGFP为报告基因,以流式细胞检测(flow cytometry ,FCM)和荧光显微照相为研究手段,比较AAV2-eGFP、AAV2/1-eGFP、Ad5-eGFP和Ad5/F35-eGFP对DFs的转染效率,探讨了四种病毒载体转染DFs的适合条件,同时利用MTT法研究四种病毒载体转染后DFs的增殖性能,希望筛选出对DFs嗜性较高的载体,同时也进一步为成纤维细胞用于基因治疗的规模化生产应用提供实验和理论依据。
AAV2-eGFP和AAV2/1-eGFP分别按照转染倍数MOI为104、105、106转染DFs,Ad5-eGFP和Ad5/F35-eGFP按照感染复数MOI为10、25、50、100转染DFs,转染后24h用流式细胞检测转染效率,荧光显微镜观察转染后的荧光强度,MTT检测两者对DFs增殖的影响。实验结果如下:
AAV2/1-eGFP对DFs无感染能力,MOI 105时AAV2转染DFs 24h后转染效率可达到(22.16+5.59)%,表明AAV2转染DFs可以有效获得皮肤基因修饰细胞,进一步用于基因治疗和基因修饰。
Ad5-eGFP和Ad5/F35-eGFP在MOI为10、25、50、100时转染效率随着MOI升高而呈上升趋势,MOI 100时,Ad5/F35-eGFP转染DFs24h后转染效率可达到90%左右,Ad5 -eGFP达到70%左右。
综合实验结果分析,结论如下:
Ad5/F35是四种载体中转染效率最高的载体,最适合实际生产应用和临床使用。AAV2载体能成功转染DFs,获得基因修饰细胞,本文所选的两种AAV载体不能解决转染效率低的问题。
Gene therapy is the introduction of genetic material into cells for therapeutic purposes. There are three elements in gene therapy. They are vector, target cells and target gene.
Dermal fibroblasts(DFs)are are convenient to obtain and prolific and hard to invert. These characteristics demonstrate that DFs are proper target cells which can be widely used in both gene therapy and gene modification.
Recent findings suggest that major breakthroughs in low transfection rate for gene therapy are imminent, thus one of the key points is to use proper vector for gene delivery. The main vectors used in gene therapy include viral vectors and non-viral vectors. As viral vector, RV (retrovirus), Ad (Adenovirus), AAV (Adeno-associated virus) and HSV (herper simplex virus) have been used in clinic which is authorized by Food And Drug Administrtion (FDA) in USA. AAV vectors are prevalent for its stable transfection and can cause almost no pathogen reactions which make it prevalent in gene therapy especially for long-term expression. On the other hand, Adenoviral vectors can easily be obtained at high titers, thus Adenoviral vectors have been widely applied in gene therapy and gene functional study. On the basis of recent study, we investigate the transfection rate of AAV2 and AAV2/1 which inserted the ITRs of AAV2 on the AAV1 as well as Ad5 and Ad5/F35 which is integrated by the fiber nob of Ad35 and the Ad5 vector at different MOIs. The improved vectors may change tissue Addiction in gene delivery.
In this investigation, DFs was isolated from human dermal skin which is authorized by related department and primarily cultured. AAV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblasts (DFs) at MOI ranging from 1. 0×104 to 1.0×106v·g/ cell .Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. Two AV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblast at MOI 10, 25, 50 and 100.Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. The killing effect of the four viruses on infective DFs was assayed respectively by MTT. The assays demonstrated following results:
AAV2/1 has no efficiency when transfection DFs. The rate of AAV2 mediated transfection at MOI 105 referred to a peak of (22.16+5.59)% which demonstrated we can obtain gene transfer cells to apply in gene therapy and gene modification using AAV2.
Transfection efficiency of Ad5-eGFP and Ad5/F35-eGFP were increased as MOI increased. At MOI 100, the rate of Ad5/F35 mediated transfection is about 90% whereas about 70% of Ad5
The results demonstrate Ad5/F35 is the best vetor during the four vectors we investigated and we can obtain gene modification cells for gene therapy using AAV2 with low transfectiong efficiency which means the low efficiency remain a problem on AAV.
皮肤成纤维细胞(dermal fibroblasts,DFs)获取方便,可以在体外大量扩增,容易进行遗传改造,并且具有容易移植和不易转化的优点,因此DFs成为目前基因治疗应用较为广泛的靶细胞,本论文亦将此细胞作为研究对象。
目前,基因治疗面临的瓶颈问题在于转染效率低下,而决定基因治疗转染效率的关键点之一就在于载体的选择。目前基因治疗所应用的载体主要包括病毒载体和非病毒载体,其中应用最多的是病毒载体,包括逆转录病毒,腺病毒,腺相关病毒,疱疹病毒和慢病毒[2],其中前四种已经被美国FDA批准应用于临床。在人类基因治疗常用的病毒载体中,腺相关病毒( Adeno-associated viruses, AAV)的安全优势以及长效表达的优点[3-4]使其成为了基因稳定转染的宠儿。而腺病毒(Adenovirus, Ad)载体则因具有感染效率和外源基因表达水平高、高滴度制备较简单等特点而被广泛用于基因治疗研究和临床试验中[5-6]。因此,本论文选择AAV和Ad为研究对象。由于病毒载体的衣壳蛋白是决定其对靶细胞嗜性的主要因素,进行衣壳蛋白改造后的载体有望改变载体亲嗜性而提高转染效率。我们在选用常规使用的AAV2和Ad5的基础上,还选用了将AAV2的ITRs包被进入AAV1的衣壳蛋白形成的嵌合型载体——AAV2/1,以及将Ad35载体的纤突整合到Ad5载体上形成的Ad5/F35作为研究对象,通过比较他们的转染效率来筛选适合DFs的载体。
本学位论文以eGFP为报告基因,以流式细胞检测(flow cytometry ,FCM)和荧光显微照相为研究手段,比较AAV2-eGFP、AAV2/1-eGFP、Ad5-eGFP和Ad5/F35-eGFP对DFs的转染效率,探讨了四种病毒载体转染DFs的适合条件,同时利用MTT法研究四种病毒载体转染后DFs的增殖性能,希望筛选出对DFs嗜性较高的载体,同时也进一步为成纤维细胞用于基因治疗的规模化生产应用提供实验和理论依据。
AAV2-eGFP和AAV2/1-eGFP分别按照转染倍数MOI为104、105、106转染DFs,Ad5-eGFP和Ad5/F35-eGFP按照感染复数MOI为10、25、50、100转染DFs,转染后24h用流式细胞检测转染效率,荧光显微镜观察转染后的荧光强度,MTT检测两者对DFs增殖的影响。实验结果如下:
AAV2/1-eGFP对DFs无感染能力,MOI 105时AAV2转染DFs 24h后转染效率可达到(22.16+5.59)%,表明AAV2转染DFs可以有效获得皮肤基因修饰细胞,进一步用于基因治疗和基因修饰。
Ad5-eGFP和Ad5/F35-eGFP在MOI为10、25、50、100时转染效率随着MOI升高而呈上升趋势,MOI 100时,Ad5/F35-eGFP转染DFs24h后转染效率可达到90%左右,Ad5 -eGFP达到70%左右。
综合实验结果分析,结论如下:
Ad5/F35是四种载体中转染效率最高的载体,最适合实际生产应用和临床使用。AAV2载体能成功转染DFs,获得基因修饰细胞,本文所选的两种AAV载体不能解决转染效率低的问题。
Gene therapy is the introduction of genetic material into cells for therapeutic purposes. There are three elements in gene therapy. They are vector, target cells and target gene.
Dermal fibroblasts(DFs)are are convenient to obtain and prolific and hard to invert. These characteristics demonstrate that DFs are proper target cells which can be widely used in both gene therapy and gene modification.
Recent findings suggest that major breakthroughs in low transfection rate for gene therapy are imminent, thus one of the key points is to use proper vector for gene delivery. The main vectors used in gene therapy include viral vectors and non-viral vectors. As viral vector, RV (retrovirus), Ad (Adenovirus), AAV (Adeno-associated virus) and HSV (herper simplex virus) have been used in clinic which is authorized by Food And Drug Administrtion (FDA) in USA. AAV vectors are prevalent for its stable transfection and can cause almost no pathogen reactions which make it prevalent in gene therapy especially for long-term expression. On the other hand, Adenoviral vectors can easily be obtained at high titers, thus Adenoviral vectors have been widely applied in gene therapy and gene functional study. On the basis of recent study, we investigate the transfection rate of AAV2 and AAV2/1 which inserted the ITRs of AAV2 on the AAV1 as well as Ad5 and Ad5/F35 which is integrated by the fiber nob of Ad35 and the Ad5 vector at different MOIs. The improved vectors may change tissue Addiction in gene delivery.
In this investigation, DFs was isolated from human dermal skin which is authorized by related department and primarily cultured. AAV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblasts (DFs) at MOI ranging from 1. 0×104 to 1.0×106v·g/ cell .Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. Two AV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblast at MOI 10, 25, 50 and 100.Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. The killing effect of the four viruses on infective DFs was assayed respectively by MTT. The assays demonstrated following results:
AAV2/1 has no efficiency when transfection DFs. The rate of AAV2 mediated transfection at MOI 105 referred to a peak of (22.16+5.59)% which demonstrated we can obtain gene transfer cells to apply in gene therapy and gene modification using AAV2.
Transfection efficiency of Ad5-eGFP and Ad5/F35-eGFP were increased as MOI increased. At MOI 100, the rate of Ad5/F35 mediated transfection is about 90% whereas about 70% of Ad5
The results demonstrate Ad5/F35 is the best vetor during the four vectors we investigated and we can obtain gene modification cells for gene therapy using AAV2 with low transfectiong efficiency which means the low efficiency remain a problem on AAV.
引文
1 Mi Ae Cho, Parham Yashar, Suk Kyoung Kim et al. HoxD10 gene delivery using Adenovirus/Adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells. Biochemical and Biophysical Research Communications, 2008(371):371-374
2 Jeffrey M. Isner. Myocardial gene therapy. Nature 2002(415), 234 - 239 insight
3 Meyers C, Mane M, Kokorina N, et al. Ubiquitous human Adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 2000; 272(2):338-46.
4 Buning H, Braun-Falco M, Hallek M. Progress in the use of Adeno-associated viral vectors for gene therapy. Cells Tissues Organs 2004; 177(3):139-50.
5 Mavilio F, Pellegrini G, Ferrari S, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006; 12(12):1397-402.
6 St. George JA. Gene therapy progress and prospects: Adenoviral vectors. Gene Ther 2003; 10(14):1135-41.
7 Yasushi K, Katsuto T, Yasufumi K. Cutaneous gene delivery Journal of Dermatological Science, 2008(50):87-98
8 Chachques JC, Salanson-Lajos C, Lajos P, et al. Cellular cardiomyoplasty for myocardial regeneration. Asian Cardiovasc Thorac Ann, 2005, 13(3): 287 - 296.
9 Bettina Burnworth, Susanne Arendt, Sonja Muffler, et al The multi-step process of human skin carcinogenesis: A role for p53, cyclin D1, hTERT, p16, and TSP-1. European Journal of Cell Biology, 2007( 86): 763-780
10 Kiran MS, Viji RI, Sameer Kumar VB, et al. Modulation of angiogenic factors by ursolic acid. Biochemical and Biophysical Research Communications, 2008(371): 556-560
11 Mandeep Singh Virk, Augustine Conduah, Sang-Hyun Park, et al. Influence of short-term Adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008(42): 921-931
12 Meyers C, Mane M, Kokorina N, Alam S, Hermonat PL.Ubiquitous human Adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 2000; 272(2):338-46.
13 Mark A.Kay, Looking into the safety of AAV vectors. Nature. 2003(424): 94305-95208
14 Bartlett J S, Wilcher R, Samulski R J. Infectious entry pathway of Adeno-associated virus and Adeno-associated virus vectors. Virol,2000, 74(6):2777-2785
15杨晓慧,孙葆忱,腺相关病毒介导绿色荧光蛋白基因对体外培养IPE细胞的转染及毒性.眼科新进展.2003,23(5):332-334
16 E.F. Stavrou, E.P. Papapetrou, A. Gianakopoulos et al. Comparative study of different promoters driving eGFP for use in episomal gene transfer in CD34+ cells[J]Blood Cells, Molecules, and Diseases, 2008,3-4( 40):286-287
17蔡文琴.现代实用细胞与分子生物学实验技术.北京:人民军医出版社,2003 : 489 - 498.
18储庆何海丽基因转移技术及其在牙周组织再生中的应用国外医学口腔医学分册2004,31(4):256-258
19 Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 72(5259):263-7.
20 Ortiz-Urda S, Thyagarajan B, Keene DR, et al. Stable nonviral genetic correction of inherited human skin disease. Nat Med 2002; 8(10):1166-70.
21 Ortiz-Urda S, Lin Q, Yant SR, Keene D, Kay MA, Khavari PA.Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer. Gene Ther 2003;10(13):1099-1104.
1 Krueger G G, Jorgensen C M, Petersen M J, et al. Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo. Human Gene Therapy, 1997. 8(5):523-532.
2 Chen M, Kasahara N, Keene DR, et al. Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa. Nat Genet 2002, 32(4):670-5.
3 Ortiz-Urda S, Lin Q, Green CL, Keene DR, Marinkovich MP,Khavari PA. Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue.J Clin Invest 2003, 111(2):251-5.
4 Woodley DT, Krueger GG, Jorgensen CM, et al. Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone. J Invest Dermatol 2003, 121(5):1021-8.
5 Woodley DT, Keene DR, Atha T, et al. Introdermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo. Mol Ther 2004;10(2):318-26.
6 Goto M, Sawamura D, Ito K, et al. Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa. J Invest Dermatol 2006;126(4):766-72.
7 Carreau M, Quilliet X, Eveno E, et al. Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients. Hum Gene Ther 1995;6(10):1307-15.
8 Zeng L, Quilliet X, Chevallier-Lagente O, Eveno E, Sarasin A,Mezzina M. Retrovirus-mediated gene transfer corrects DNA epair defect of xeroderma pigmentosum cells of complementation groups A, B and C. Gene Ther 1997;4(10):1077-84.
9 Marchetto MC, Muotri AR, Burns DK, Friedberg EC, Menck CF.Gene transduction in skin cells: preventing cancer in xeroderma pigmentosum mice. Proc Natl AcAd Sci USA 2004;101(51):17759-64.
10 Marchetto MC, Correa RG, Menck CF, Muotri AR. Functional lentiviral vectors for xeroderma pigmentosum gene therapy.J Biotechnol 2006;126(4):424-30.
11 Meng X, Sawamura D, Tamai K, HanAda K, Ishida H, Hashimoto I. Keratinocyte genetherapy for systemic diseases.Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin. J Clin Invest 1998;101(6):1462-7.
12 Beak SC, Lin Q, Robbins PB, Fan H, Khavari PA. Sustainable systemic delivery via a single injection of lentivirus into human skin tissue. Hum Gene Ther 2001;12(12):1551-8.
13 Christensen R, Guttler F, Jensen TG. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria. Mol Genet Metab 2002;76(4):313-8.
14 Meng X, Sawamura D, Ina S, Tamai K, HanAda K, Hashimoto I.Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducingcytokine genes into skin. Exp Dermatol 2002;11(5):456-61.
15 Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious Adverse event after successful gene therapy for X-linked severe combined immuno deficiency. N Engl J Med 2003;348(3):255-6.
16 Mavilio F, Pellegrini G, Ferrari S, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006;12(12):1397-402.
17 St. George JA. Gene therapy progress and prospects: Adenoviral vectors. Gene Ther 2003;10(14):1135-41.
18 Huber M, Limat A, Wagner E, Hohl D. Efficient in vitro transfection of human keratinocytes with an Adenovirusenhanced receptor-mediated system. J Invest Dermatol 2000;114(4):661-6.
19 ONO I , YAMASHITA T , HIDA T , et al. Local Adminis-tration of hepatocyte growth factor gene enhances the regen-eration of dermis in acute incisional wounds [J] . J Surg Res 2004 ,120 (1) :47255.
20 HA X , L I Y, LAO M ,et al. Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by Adenovirus2mediated gene transfer [J] Chin Med J ( Engl) , 2003 ,116 (7) :102921033.
21 Krueger G G, Jorgensen C M, Petersen M J, et al. Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo. Human Gene Therapy, 1997. 8(5):523-532.
22. Wei J F, Wei F S, Samulski R J, et al. Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an Adeno-associated virus vector. Gene Ther, 1994. 1(4):261-8.
23. Sakamoto H, Ochiya T, Sato Y, et al. Adenovirus-mediated transfer of the HST-1 (FGF4) gene induces increased levels of platelet count in vivo. Proc Natl AcAd Sci U S A, 1994. 91(26):12368-72.
24. Lucidi V, Rosati P, Bella S, et al. [Clinical heterogeneity at the diagnosis in a cystic fibrosis population]. Pediatr Med Chir, 1995. 17(1):57-9.
25 La Gamma EF, Weisinger G, Lenn NJ, et al. Genetically modified primary astrocytes as vehicles for gene therapy in the brain. Cell transplant. 1993; 2: 207-214
26. Ray J, Hogg J, Beutler A S, et al. Expression of biologically active basic fibroblast growth factor by genetically modified rat primary skin fibroblasts. J Neurochem, 1995. 64(2):503-13.
27. Tuszynski M H, Roberts J, Senut M C, et al. Gene therapy in the Adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. Gene Ther, 1996. 3(4):305-14.
28. Rossner S, Yu J, Pizzo D, et al. Effects of intraventricular transplantation of NGF-secreting cells on cholinergic basal forebrain neurons after partial immunolesion. J Neurosci Res, 1996. 45(1):40-56.
29. Geschwind M D, Hartnick C J, Liu W, et al. Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. Hum Gene Ther, 1996. 7(2):173-82.
30. Ijichi A, Noel F, Sakuma S, et al. Ex vivo gene delivery of platelet-derived growth factor increases 0-2A progenitors in Adult rat spinal cord. Gene Ther, 1996. 3(5):389-95.
31 Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the hair follicle. Nat Med 1995;1(7):705-6
32 Kaneda Y, Iwai K, Uchida T. Increased expression of DNA cointroduced with nuclear protein in Adult rat liver. Science 1989; 43(4889):375-8.
33 Hayashi SI, Morishita R, Aoki M, et al. In vivo transfer of gene and oligo-eoxynucleotides into skin of fetal rats by incubation in amniotic fluid. Gene Ther 1996;3(10):878—85.
34 Parveen Z, Krupetsky A, EngelstAdter M, etal. Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells. Nat Biotechnol, 2000, 18(6): 623-629.
35 Steidl S, Stitz J, Schmitt I, et al. Coreceptor Switch of [MLV (SIVagm)] pseudotype vectors by V3-loop exchange. Virology,2002, 300(2): 205-216.
36 John Wiley & Sons Ltd. Adenoviral Vectors.Gene Therapy Technologies, Applications and Regulations.1999.
37金天明,武迎红.腺病毒及腺病毒载体研究进展.天津农学院学报.2007, 14(2):48-51.
38 Dragomira Majhen, Andreja Ambriovi′c-Ristov. Adenoviral vectors-How to use them in cancer gene therapy?. Virus Research.2006, 119:121-133.
39 X. Zhang, W.T. Godbey. Viral vectors for gene delivery in tissue engineering.Advanced Drug Delivery Reviews.2006, 58:515-534.
40 Bergelson J M, Cunningham J A, Droguett G,et al.Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and5. Science.1997, 275 (5304):1320-1323.
41 Meier, O., Boucke, K., Hammer, S.V., et al. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J. Cell Biol. 2002, 158:1119-1131.
42 Meier, O, Greber, UF. Adenovirus endocytosis. J. Gene Med. 2004, 6:S152-S163.
43 Wang K, Huang S, Kapoor M A, et a1. Adenovirus internalization and infection require dynamin.J Virol. 1998, 72: 3455-3458.
44 W.C. Russell. Update on adenovirus and its vectors. Gen.Virol. 2000, 81: 2573-2604.
45 Isabelle Nadeau, Amine Kamen. Production of adenovirus vector for gene therapy. Biotechnology Advances. 2003, 20: 475-489.
46 Haisma HJ, Pinedo HM, van Rijswijk A, et al. Tumor-specific gene transfer via an adenoviral vector targeted to the pancarcinoma antigen EpCAM. Gene Ther, 1999, 6: 1469-1474
47 Dmitriev I, Kraanykh V, Millerc R, et al. An adenovirus vector with genetically modified fibers demonstrats expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Vrial, 1998, 72: 9706-9713
48 Schiedner G, Morral N, Parks RJ, et al. Genomic DNA transfer with a high–capacity Adenovius vector results in improved in vivo gene expression and decreased toxicity. Nat Genet, 1998, 18(2): 180-183.
49 Sandig V, Youil R, Bett AJ, et al. Optimization of the helper-dependent Adenovirus system for production and potency in vivo. Proc Natl AcAd Sci USA, 2000, 97(3): 1002-1007.
50 Schoggins JW, Gall JG, Falck-Pedersen E. Subgroup B and f fiber chimeras eliminate normal Adenovirus type 5 vector transduction in vitro and in vivo. J Virol, 2003, 77(2): 1039-1048.
51 Ichrikawa T, Chiocca EA. Comparative analysis of transgene delivery and expression in tumor sinoculated with a replication-conditional or defective viral vector. Cancer Res, 2001, 61(14): 5336-5339.
52 Zabner J, Couture LA, Gregory RJ, et al. Adenovirus-mediated transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell, 1993, 75: 206-216.
53 Batshaw ML, Wilson JM, Yudkoff M, et al. Recombinant adenovirus gene transfer in adults with partial orinthine transcarbamylase deficiency(OTCD). Hum Gene Ther, 1999, 10: 2419-2437.
54 Lai CM, Lai YKY, Rakocay PE, Adenovirus and adeno-associated virus vectors. DNA Cell Biol, 2002, 21:895-913
55 Van Breukelen B, Kanellopoulos PN, Turker P, etal. The information of aflexible DNA unwinding and adenovirus DNA chain elongation. J Biol Chem, 2000, 275: 897-903
56 Yan Z, Zak R, Zhang Y, et al. Inverted terminal repeat sequences are important for inter molecular recombination and circularization of adeno-associated virus genomes. J Virol, 2005, 79 (1) : 364-379
57 Ding W, Zhang L, Yan Z, et al. Intracellular trafficking of adeno-associated viral vectors.GeneTherapy, 2005, 12 (11) :873-880
58 Xiao W, Chirmule N, Berta S C, et al. Gene therapy vectors based on adeno- associated virus type 1. J Virol, 1999, 73 (5) : 3994-4003
59 Handa A,Muramatsu S,Qiu J, etal. Adeno-associatedvirus (AAV )-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2 based vectors. J GenVirol,2000, 81 (8): 2077-2084
60 Zabner J, Seiler M, Walters R, etal. Adeno-associatedvirustype5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol, 2000,74 (8): 3852-3858
61 Davidson BL, Stein CS, Heth JA, et al. Recombinant adeno-associated virus type2, 4 and 5 vectors: transduction of variant cell type sand regionsin themammalian central nervous system. Proc Natl Acad Sci USA, 2000, 97 (7): 3432-3428
62 Wang Z,Zhu T,Qiao C, etal. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol, 2005, 23(3): 321-328
63 TR et al Gene Therapy 1995; 2: 29
64 Mammounas M et al Gene Therapy 1995; 2: 429
65 HermensW T et al Hum Gene Ther 1999; 10: 1885
66 Du L, Kido M, Lee DV, etal. Differential myocardial gene deliveryby recombination serotype-specific adeno-associated viral vector. Mol Ther,2004,10(3): 604-608
67 SanligouAD, Karacay B, Benson PK, etal. Novel approaches to augment adeno-associated virustype-2 endocytosis and transduction.Virus Res,2004,104(1):51-59
68 Sanligou S,Benson PK,Yang Z,etal. Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by racl and phosphatidylinositol-3 kinase activation. J Viral, 2000, 74, 9: 134-196
69 Zhong L, Li W,Yang Z, etal. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 in vivo. Gene Ther, 2004, 11(14):1165-9
69 Russell DW, Miller AD,Alexander IE. Adeno-associated virus vectors preferentially transduce cells in Sphase. Proc NatlA cad SciUSA,1994, 91 (19):8915-8919
70 Flotte TR et al. PhaseⅠtrail of intranasaland endobronchial administration of a recombinant adeno -associated virus serotype2 ( rAAV2) -CFTR vector in adult cystic fibrosis patients: a two-part clinical study. Hum Gene Ther 2003; 14: 1079-1088
71 Wagner JA et al. A phaseⅡ, double-blinded, randomized, placebo-controlled clinical trail of tgAAVCF using maxillary sinus delivery inpatients with cystic frosis with antrostomies. Hum Gene Ther 2002; 13: 1349-1359
72 Aitken ML et al. A phaseⅠstudy of aerosolized administration of tgAAVCF to Cysticfbrosis subjects with mild lung disease. HumGeneTher 2001;12:1907～1916
73 Manno CS et al. AAV-mediated factorⅨgene transfer to skeletal muscle in patients with hemaphilia B. Blood 2003; 101: 2963-2972
74 Takuji koro, Korchi Miyake et al. Adeno-Associated Viral Vector-Mediated Expressionof Endostatin Inhibits Tumor Growth and Metastasis in an Orthotropic Pancreatic Cancer Model in Hamsters. Cancer Research 2004;64:7486-7490
75 Wagner JA, Reynolds T, Moran ML, et al. Efficient and persistant gene transfer of AAV-CFTR in maxillary sinus. Lancet, 1998, 351: 1702-1703.
76 Asfour B, Baba HA, Scheld HH, et al. Uniform long-term gene expression using Adeno-associatedvirus(AAV) by exvivo recirculation in rat-cardiac isografts. Thorac Cardiovasc Surg, 2002, 50(6): 347-350.
77 Nicklin SA, Baker AH. Tropism-modified Adenoviral and Adeno-associated viral vectors for gene therapy. Curr Gene Ther, 2002, 2(3): 273-293.
78 Ponnazhagan S, Mahendra G, Kumar S, et al. Conjugate-based targeting of recombinant Adeno-associated virus type 2 vectors by using avidin-linked ligands. J Virol, 2002, 76(24): 12900-12907.
79 Indraccolo S, Habeler W, Tisato V, et al. Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors. Cancer Res, 2002, 62(21): 6099-6107.
80 Lim FY, Kobinger GP, Weiner DJ, et al. Human fetal trachea-scid mouse xenografts: Efficacy of vesiculars to matitis virus-G pseudo typed lentiviral-mediated gene transfer. J Pediatr Surg,2003, 38(6): 834-839.
81 Olsen JC. EIAV, CAEV and other lentivirus vector systems. So-mat Cell Mol Genet, 2001, 26(126): 131-145.
82 Fukuda Y, Yamamura J, Uwano T, et al. Regulated transgene delivery by ganciclovir in the brain without physiological alterations by alive attenuated herpes simplex virus vector. Neurosci Res, 2003, 45(2): 233-241.
83 Stevenson AF, Frolova Jones E, Hall KT, et al. A herpesvirus saim iri-based gene therapy vector with potential foruse in cancer immuno therapy. Cancer Gene Ther, 2000, 7(7): 1077-1085.
84 Moriuchi S, Krisky DM, Marconi PC, et al.HSV vector cytotoxicity is inverselycorrelated with effective TK/GCV suicide gene therapy of rat gliosarcoma. Gene Ther, 2000, 7(17): 1483-1490.
85 Wu H, Dmitriev I, Kashentseva E, et al.Construction and characterization of Adenovirus serotype 5 packaged by serotype 3 hexon. J Virol, 2002, 76(24):12775-12782.
86 Goncalves M A, vander Velde I, Janssen J M, etal. Efficient gene ration and amplification of high-capacity Adeno-associatedvirus/Adenovirus hybrid vectors. J Virol, 2002,76(21):10734-10744.
87 Murphy SJ, ChongH, Bell S, etal. Novel integrating Adenoviral/ retroviral hybrid vector for gene therapy. HumGene Ther,2002,13(6):745-760.
88 WangY, Camp SM, Niwano M, et al. Herpes simplex virus type 1/ Adeno-associat edvirus rep (+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration. J Virol, 2002, 76(14): 7150-7162.
2 Jeffrey M. Isner. Myocardial gene therapy. Nature 2002(415), 234 - 239 insight
3 Meyers C, Mane M, Kokorina N, et al. Ubiquitous human Adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 2000; 272(2):338-46.
4 Buning H, Braun-Falco M, Hallek M. Progress in the use of Adeno-associated viral vectors for gene therapy. Cells Tissues Organs 2004; 177(3):139-50.
5 Mavilio F, Pellegrini G, Ferrari S, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006; 12(12):1397-402.
6 St. George JA. Gene therapy progress and prospects: Adenoviral vectors. Gene Ther 2003; 10(14):1135-41.
7 Yasushi K, Katsuto T, Yasufumi K. Cutaneous gene delivery Journal of Dermatological Science, 2008(50):87-98
8 Chachques JC, Salanson-Lajos C, Lajos P, et al. Cellular cardiomyoplasty for myocardial regeneration. Asian Cardiovasc Thorac Ann, 2005, 13(3): 287 - 296.
9 Bettina Burnworth, Susanne Arendt, Sonja Muffler, et al The multi-step process of human skin carcinogenesis: A role for p53, cyclin D1, hTERT, p16, and TSP-1. European Journal of Cell Biology, 2007( 86): 763-780
10 Kiran MS, Viji RI, Sameer Kumar VB, et al. Modulation of angiogenic factors by ursolic acid. Biochemical and Biophysical Research Communications, 2008(371): 556-560
11 Mandeep Singh Virk, Augustine Conduah, Sang-Hyun Park, et al. Influence of short-term Adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone, 2008(42): 921-931
12 Meyers C, Mane M, Kokorina N, Alam S, Hermonat PL.Ubiquitous human Adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 2000; 272(2):338-46.
13 Mark A.Kay, Looking into the safety of AAV vectors. Nature. 2003(424): 94305-95208
14 Bartlett J S, Wilcher R, Samulski R J. Infectious entry pathway of Adeno-associated virus and Adeno-associated virus vectors. Virol,2000, 74(6):2777-2785
15杨晓慧,孙葆忱,腺相关病毒介导绿色荧光蛋白基因对体外培养IPE细胞的转染及毒性.眼科新进展.2003,23(5):332-334
16 E.F. Stavrou, E.P. Papapetrou, A. Gianakopoulos et al. Comparative study of different promoters driving eGFP for use in episomal gene transfer in CD34+ cells[J]Blood Cells, Molecules, and Diseases, 2008,3-4( 40):286-287
17蔡文琴.现代实用细胞与分子生物学实验技术.北京:人民军医出版社,2003 : 489 - 498.
18储庆何海丽基因转移技术及其在牙周组织再生中的应用国外医学口腔医学分册2004,31(4):256-258
19 Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 72(5259):263-7.
20 Ortiz-Urda S, Thyagarajan B, Keene DR, et al. Stable nonviral genetic correction of inherited human skin disease. Nat Med 2002; 8(10):1166-70.
21 Ortiz-Urda S, Lin Q, Yant SR, Keene D, Kay MA, Khavari PA.Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer. Gene Ther 2003;10(13):1099-1104.
1 Krueger G G, Jorgensen C M, Petersen M J, et al. Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo. Human Gene Therapy, 1997. 8(5):523-532.
2 Chen M, Kasahara N, Keene DR, et al. Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa. Nat Genet 2002, 32(4):670-5.
3 Ortiz-Urda S, Lin Q, Green CL, Keene DR, Marinkovich MP,Khavari PA. Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue.J Clin Invest 2003, 111(2):251-5.
4 Woodley DT, Krueger GG, Jorgensen CM, et al. Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone. J Invest Dermatol 2003, 121(5):1021-8.
5 Woodley DT, Keene DR, Atha T, et al. Introdermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo. Mol Ther 2004;10(2):318-26.
6 Goto M, Sawamura D, Ito K, et al. Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa. J Invest Dermatol 2006;126(4):766-72.
7 Carreau M, Quilliet X, Eveno E, et al. Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients. Hum Gene Ther 1995;6(10):1307-15.
8 Zeng L, Quilliet X, Chevallier-Lagente O, Eveno E, Sarasin A,Mezzina M. Retrovirus-mediated gene transfer corrects DNA epair defect of xeroderma pigmentosum cells of complementation groups A, B and C. Gene Ther 1997;4(10):1077-84.
9 Marchetto MC, Muotri AR, Burns DK, Friedberg EC, Menck CF.Gene transduction in skin cells: preventing cancer in xeroderma pigmentosum mice. Proc Natl AcAd Sci USA 2004;101(51):17759-64.
10 Marchetto MC, Correa RG, Menck CF, Muotri AR. Functional lentiviral vectors for xeroderma pigmentosum gene therapy.J Biotechnol 2006;126(4):424-30.
11 Meng X, Sawamura D, Tamai K, HanAda K, Ishida H, Hashimoto I. Keratinocyte genetherapy for systemic diseases.Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin. J Clin Invest 1998;101(6):1462-7.
12 Beak SC, Lin Q, Robbins PB, Fan H, Khavari PA. Sustainable systemic delivery via a single injection of lentivirus into human skin tissue. Hum Gene Ther 2001;12(12):1551-8.
13 Christensen R, Guttler F, Jensen TG. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria. Mol Genet Metab 2002;76(4):313-8.
14 Meng X, Sawamura D, Ina S, Tamai K, HanAda K, Hashimoto I.Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducingcytokine genes into skin. Exp Dermatol 2002;11(5):456-61.
15 Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious Adverse event after successful gene therapy for X-linked severe combined immuno deficiency. N Engl J Med 2003;348(3):255-6.
16 Mavilio F, Pellegrini G, Ferrari S, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006;12(12):1397-402.
17 St. George JA. Gene therapy progress and prospects: Adenoviral vectors. Gene Ther 2003;10(14):1135-41.
18 Huber M, Limat A, Wagner E, Hohl D. Efficient in vitro transfection of human keratinocytes with an Adenovirusenhanced receptor-mediated system. J Invest Dermatol 2000;114(4):661-6.
19 ONO I , YAMASHITA T , HIDA T , et al. Local Adminis-tration of hepatocyte growth factor gene enhances the regen-eration of dermis in acute incisional wounds [J] . J Surg Res 2004 ,120 (1) :47255.
20 HA X , L I Y, LAO M ,et al. Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by Adenovirus2mediated gene transfer [J] Chin Med J ( Engl) , 2003 ,116 (7) :102921033.
21 Krueger G G, Jorgensen C M, Petersen M J, et al. Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo. Human Gene Therapy, 1997. 8(5):523-532.
22. Wei J F, Wei F S, Samulski R J, et al. Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an Adeno-associated virus vector. Gene Ther, 1994. 1(4):261-8.
23. Sakamoto H, Ochiya T, Sato Y, et al. Adenovirus-mediated transfer of the HST-1 (FGF4) gene induces increased levels of platelet count in vivo. Proc Natl AcAd Sci U S A, 1994. 91(26):12368-72.
24. Lucidi V, Rosati P, Bella S, et al. [Clinical heterogeneity at the diagnosis in a cystic fibrosis population]. Pediatr Med Chir, 1995. 17(1):57-9.
25 La Gamma EF, Weisinger G, Lenn NJ, et al. Genetically modified primary astrocytes as vehicles for gene therapy in the brain. Cell transplant. 1993; 2: 207-214
26. Ray J, Hogg J, Beutler A S, et al. Expression of biologically active basic fibroblast growth factor by genetically modified rat primary skin fibroblasts. J Neurochem, 1995. 64(2):503-13.
27. Tuszynski M H, Roberts J, Senut M C, et al. Gene therapy in the Adult primate brain: intraparenchymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. Gene Ther, 1996. 3(4):305-14.
28. Rossner S, Yu J, Pizzo D, et al. Effects of intraventricular transplantation of NGF-secreting cells on cholinergic basal forebrain neurons after partial immunolesion. J Neurosci Res, 1996. 45(1):40-56.
29. Geschwind M D, Hartnick C J, Liu W, et al. Defective HSV-1 vector expressing BDNF in auditory ganglia elicits neurite outgrowth: model for treatment of neuron loss following cochlear degeneration. Hum Gene Ther, 1996. 7(2):173-82.
30. Ijichi A, Noel F, Sakuma S, et al. Ex vivo gene delivery of platelet-derived growth factor increases 0-2A progenitors in Adult rat spinal cord. Gene Ther, 1996. 3(5):389-95.
31 Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the hair follicle. Nat Med 1995;1(7):705-6
32 Kaneda Y, Iwai K, Uchida T. Increased expression of DNA cointroduced with nuclear protein in Adult rat liver. Science 1989; 43(4889):375-8.
33 Hayashi SI, Morishita R, Aoki M, et al. In vivo transfer of gene and oligo-eoxynucleotides into skin of fetal rats by incubation in amniotic fluid. Gene Ther 1996;3(10):878—85.
34 Parveen Z, Krupetsky A, EngelstAdter M, etal. Spleen necrosis virus-derived C-type retroviral vectors for gene transfer to quiescent cells. Nat Biotechnol, 2000, 18(6): 623-629.
35 Steidl S, Stitz J, Schmitt I, et al. Coreceptor Switch of [MLV (SIVagm)] pseudotype vectors by V3-loop exchange. Virology,2002, 300(2): 205-216.
36 John Wiley & Sons Ltd. Adenoviral Vectors.Gene Therapy Technologies, Applications and Regulations.1999.
37金天明,武迎红.腺病毒及腺病毒载体研究进展.天津农学院学报.2007, 14(2):48-51.
38 Dragomira Majhen, Andreja Ambriovi′c-Ristov. Adenoviral vectors-How to use them in cancer gene therapy?. Virus Research.2006, 119:121-133.
39 X. Zhang, W.T. Godbey. Viral vectors for gene delivery in tissue engineering.Advanced Drug Delivery Reviews.2006, 58:515-534.
40 Bergelson J M, Cunningham J A, Droguett G,et al.Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and5. Science.1997, 275 (5304):1320-1323.
41 Meier, O., Boucke, K., Hammer, S.V., et al. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J. Cell Biol. 2002, 158:1119-1131.
42 Meier, O, Greber, UF. Adenovirus endocytosis. J. Gene Med. 2004, 6:S152-S163.
43 Wang K, Huang S, Kapoor M A, et a1. Adenovirus internalization and infection require dynamin.J Virol. 1998, 72: 3455-3458.
44 W.C. Russell. Update on adenovirus and its vectors. Gen.Virol. 2000, 81: 2573-2604.
45 Isabelle Nadeau, Amine Kamen. Production of adenovirus vector for gene therapy. Biotechnology Advances. 2003, 20: 475-489.
46 Haisma HJ, Pinedo HM, van Rijswijk A, et al. Tumor-specific gene transfer via an adenoviral vector targeted to the pancarcinoma antigen EpCAM. Gene Ther, 1999, 6: 1469-1474
47 Dmitriev I, Kraanykh V, Millerc R, et al. An adenovirus vector with genetically modified fibers demonstrats expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Vrial, 1998, 72: 9706-9713
48 Schiedner G, Morral N, Parks RJ, et al. Genomic DNA transfer with a high–capacity Adenovius vector results in improved in vivo gene expression and decreased toxicity. Nat Genet, 1998, 18(2): 180-183.
49 Sandig V, Youil R, Bett AJ, et al. Optimization of the helper-dependent Adenovirus system for production and potency in vivo. Proc Natl AcAd Sci USA, 2000, 97(3): 1002-1007.
50 Schoggins JW, Gall JG, Falck-Pedersen E. Subgroup B and f fiber chimeras eliminate normal Adenovirus type 5 vector transduction in vitro and in vivo. J Virol, 2003, 77(2): 1039-1048.
51 Ichrikawa T, Chiocca EA. Comparative analysis of transgene delivery and expression in tumor sinoculated with a replication-conditional or defective viral vector. Cancer Res, 2001, 61(14): 5336-5339.
52 Zabner J, Couture LA, Gregory RJ, et al. Adenovirus-mediated transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell, 1993, 75: 206-216.
53 Batshaw ML, Wilson JM, Yudkoff M, et al. Recombinant adenovirus gene transfer in adults with partial orinthine transcarbamylase deficiency(OTCD). Hum Gene Ther, 1999, 10: 2419-2437.
54 Lai CM, Lai YKY, Rakocay PE, Adenovirus and adeno-associated virus vectors. DNA Cell Biol, 2002, 21:895-913
55 Van Breukelen B, Kanellopoulos PN, Turker P, etal. The information of aflexible DNA unwinding and adenovirus DNA chain elongation. J Biol Chem, 2000, 275: 897-903
56 Yan Z, Zak R, Zhang Y, et al. Inverted terminal repeat sequences are important for inter molecular recombination and circularization of adeno-associated virus genomes. J Virol, 2005, 79 (1) : 364-379
57 Ding W, Zhang L, Yan Z, et al. Intracellular trafficking of adeno-associated viral vectors.GeneTherapy, 2005, 12 (11) :873-880
58 Xiao W, Chirmule N, Berta S C, et al. Gene therapy vectors based on adeno- associated virus type 1. J Virol, 1999, 73 (5) : 3994-4003
59 Handa A,Muramatsu S,Qiu J, etal. Adeno-associatedvirus (AAV )-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2 based vectors. J GenVirol,2000, 81 (8): 2077-2084
60 Zabner J, Seiler M, Walters R, etal. Adeno-associatedvirustype5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol, 2000,74 (8): 3852-3858
61 Davidson BL, Stein CS, Heth JA, et al. Recombinant adeno-associated virus type2, 4 and 5 vectors: transduction of variant cell type sand regionsin themammalian central nervous system. Proc Natl Acad Sci USA, 2000, 97 (7): 3432-3428
62 Wang Z,Zhu T,Qiao C, etal. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol, 2005, 23(3): 321-328
63 TR et al Gene Therapy 1995; 2: 29
64 Mammounas M et al Gene Therapy 1995; 2: 429
65 HermensW T et al Hum Gene Ther 1999; 10: 1885
66 Du L, Kido M, Lee DV, etal. Differential myocardial gene deliveryby recombination serotype-specific adeno-associated viral vector. Mol Ther,2004,10(3): 604-608
67 SanligouAD, Karacay B, Benson PK, etal. Novel approaches to augment adeno-associated virustype-2 endocytosis and transduction.Virus Res,2004,104(1):51-59
68 Sanligou S,Benson PK,Yang Z,etal. Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by racl and phosphatidylinositol-3 kinase activation. J Viral, 2000, 74, 9: 134-196
69 Zhong L, Li W,Yang Z, etal. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 in vivo. Gene Ther, 2004, 11(14):1165-9
69 Russell DW, Miller AD,Alexander IE. Adeno-associated virus vectors preferentially transduce cells in Sphase. Proc NatlA cad SciUSA,1994, 91 (19):8915-8919
70 Flotte TR et al. PhaseⅠtrail of intranasaland endobronchial administration of a recombinant adeno -associated virus serotype2 ( rAAV2) -CFTR vector in adult cystic fibrosis patients: a two-part clinical study. Hum Gene Ther 2003; 14: 1079-1088
71 Wagner JA et al. A phaseⅡ, double-blinded, randomized, placebo-controlled clinical trail of tgAAVCF using maxillary sinus delivery inpatients with cystic frosis with antrostomies. Hum Gene Ther 2002; 13: 1349-1359
72 Aitken ML et al. A phaseⅠstudy of aerosolized administration of tgAAVCF to Cysticfbrosis subjects with mild lung disease. HumGeneTher 2001;12:1907～1916
73 Manno CS et al. AAV-mediated factorⅨgene transfer to skeletal muscle in patients with hemaphilia B. Blood 2003; 101: 2963-2972
74 Takuji koro, Korchi Miyake et al. Adeno-Associated Viral Vector-Mediated Expressionof Endostatin Inhibits Tumor Growth and Metastasis in an Orthotropic Pancreatic Cancer Model in Hamsters. Cancer Research 2004;64:7486-7490
75 Wagner JA, Reynolds T, Moran ML, et al. Efficient and persistant gene transfer of AAV-CFTR in maxillary sinus. Lancet, 1998, 351: 1702-1703.
76 Asfour B, Baba HA, Scheld HH, et al. Uniform long-term gene expression using Adeno-associatedvirus(AAV) by exvivo recirculation in rat-cardiac isografts. Thorac Cardiovasc Surg, 2002, 50(6): 347-350.
77 Nicklin SA, Baker AH. Tropism-modified Adenoviral and Adeno-associated viral vectors for gene therapy. Curr Gene Ther, 2002, 2(3): 273-293.
78 Ponnazhagan S, Mahendra G, Kumar S, et al. Conjugate-based targeting of recombinant Adeno-associated virus type 2 vectors by using avidin-linked ligands. J Virol, 2002, 76(24): 12900-12907.
79 Indraccolo S, Habeler W, Tisato V, et al. Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors. Cancer Res, 2002, 62(21): 6099-6107.
80 Lim FY, Kobinger GP, Weiner DJ, et al. Human fetal trachea-scid mouse xenografts: Efficacy of vesiculars to matitis virus-G pseudo typed lentiviral-mediated gene transfer. J Pediatr Surg,2003, 38(6): 834-839.
81 Olsen JC. EIAV, CAEV and other lentivirus vector systems. So-mat Cell Mol Genet, 2001, 26(126): 131-145.
82 Fukuda Y, Yamamura J, Uwano T, et al. Regulated transgene delivery by ganciclovir in the brain without physiological alterations by alive attenuated herpes simplex virus vector. Neurosci Res, 2003, 45(2): 233-241.
83 Stevenson AF, Frolova Jones E, Hall KT, et al. A herpesvirus saim iri-based gene therapy vector with potential foruse in cancer immuno therapy. Cancer Gene Ther, 2000, 7(7): 1077-1085.
84 Moriuchi S, Krisky DM, Marconi PC, et al.HSV vector cytotoxicity is inverselycorrelated with effective TK/GCV suicide gene therapy of rat gliosarcoma. Gene Ther, 2000, 7(17): 1483-1490.
85 Wu H, Dmitriev I, Kashentseva E, et al.Construction and characterization of Adenovirus serotype 5 packaged by serotype 3 hexon. J Virol, 2002, 76(24):12775-12782.
86 Goncalves M A, vander Velde I, Janssen J M, etal. Efficient gene ration and amplification of high-capacity Adeno-associatedvirus/Adenovirus hybrid vectors. J Virol, 2002,76(21):10734-10744.
87 Murphy SJ, ChongH, Bell S, etal. Novel integrating Adenoviral/ retroviral hybrid vector for gene therapy. HumGene Ther,2002,13(6):745-760.
88 WangY, Camp SM, Niwano M, et al. Herpes simplex virus type 1/ Adeno-associat edvirus rep (+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration. J Virol, 2002, 76(14): 7150-7162.