含癌胚抗原片段的重组腺相关病毒转染树突状细胞疫苗的体外杀伤作用

目的：观察研究人外周血单核细胞来源的树突状细胞（DC）转染含癌胚抗原（CEA）片段的重组腺相关病毒后所诱导的特异性T细胞对直肠癌细胞株LOVO和SW480的体外杀伤作用。方法：抽取HLA表型为A11的健康志愿者外周血，分离单核细胞，体外培养，使用含CEA片断的重组腺相关病毒转染未成熟DC，诱导特异性T细胞。检测体外培养的DC和CTL活性，并使用MTT法检测细胞毒性T细胞（CTL）对LOVO细胞的杀伤作用。结果：转染或未转染的体外培养的成熟DC高表达CD40、CD86、IL-12，诱导的细胞毒性T细胞高表达IFN-γ；转染后DC诱导特异性细胞毒性T细胞可有效识别并杀伤HLA-A11阳性的LOVO细胞。结论：重组腺相关病毒转染DC，不明显改变DC表型和刺激淋巴细胞增殖、分化功能，可诱导自体细胞毒性T细胞增殖，含CEA片断的腺相关病毒转染DC诱导自体细胞毒性T细胞对LOVO细胞有明显杀伤作用，DC疫苗可以作为直肠癌患者免疫治疗的有效补充。     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1（glucagon-like peptide-1,GLP-1）在人体内半衰期过短限制了其应用。目的：构建可表达GLP-1的重组腺伴随病毒。方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景:胰高血糖素样肽1(glucagon-like peptide-1,GLP-1)在人体内半衰期过短限制了其应用.目的:构建可表达GLP-1的重组腺伴随病毒.方法:将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒.采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞.结果与结论:重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内.免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1.     

腺相关病毒介导癌胚抗原转染树突状细胞对免疫功能的调节作用

目的:近年来,树突状细胞参与调节作用的研究较多。实验拟验证重组腺相关病毒(rAAV)介导癌胚抗原基因转染树突状细胞后其的免疫调节作用的变化。方法:实验于2006-06/2006-11在美国阿肯色大学医学院基因治疗中心完成。①实验方法:取健康人外周血(自愿捐献),采用改良Ficoll密度梯度离心的方法分离外周血单个核细胞,取贴壁细胞,分为rAAV/CEA转染组和空白对照组,均采用重组人粒细胞巨噬细胞集落刺激因子、白细胞介素4、肿瘤坏死因子-α诱导树突状细胞前体细胞成熟,将成熟树突状细胞与末梢血淋巴细胞按比例混合培养,可得到激活的细胞毒性T淋巴细胞。②实验评估:采用流式细胞仪检测树突状细胞表面标志表达,细胞毒性T淋巴细胞的免疫表型变化,细胞毒性T淋巴细胞γ-干扰素表达。采用酶联免疫吸附法(ELISA法)检测树突状细胞的白细胞介素12和白细胞介素10表达。结果:①rAAV/CEA转染树突状细胞的CD14较空白对照组降低,共刺激分子CD40、CD80、CD83、CD86表达均较空白对照组高。②成熟树突状细胞细胞因子表达中,rAAV/CEA转染组的白细胞介素12较空白对照组升高,白细胞介素10降低。③与空白对照组比较,rAAV/CEA转染组能高表达CD8 T细胞和其表型CD69,CD8/CD56的T细胞比例上调,CD25 CD4 的T细胞减少。④rAAV/CEA转染的细胞毒性T淋巴细胞表达相对较高水平的γ-干扰素,与杀伤实验结果相吻合。结论:rAAV/CEA转染树突状细胞能够激活细胞毒性T淋巴细胞的特异性杀伤作用,与树突状细胞和细胞毒性T淋巴细胞等免疫细胞表面标志变化和细胞内细胞因子水平变化相关。     

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.     

Production of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed for preclinical studies. Interest in rAAV has been driven by advances in production methods originally developed for rAAV serotype 2 vectors and expanded to include alternative serotypes. The transition to clinical trials is dependent on the development of scalable production methods of Good Manufacturing Practice-grade vectors described in this review.     

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.     

小鼠脂联素重组腺相关病毒载体的构建

目的:构建脂联素重组腺相关病毒载体,为进一步研究脂联素的功能提供基础。方法:实验于2003年在中山大学附属第二医院林百欣医学研究中心进行。以带有小鼠脂联素基因的质粒pcDNA3.0-Ad为模板,聚合酶链反应克隆脂联素基因,并将聚合酶链反应扩增产物定向克隆于pAAV-MCS载体,重组AAV-MCS-Ad质粒经双酶切和测序鉴定。随后采用磷酸钙转染法将pAAV-MCS-Ad、pAAV-RC,pAAV-Helper质粒共转染AAV-293细胞,包装得到重组腺相关病毒穴rAAV-Ad雪。回收病毒原液,采用PEG8000和氯仿纯化浓缩病毒。SDS-PAGE及透射电镜鉴定纯化的病毒,并采用Southernblot测定重组腺相关病毒的滴度。Westernblotting检测rAAV-Ad转染H4IIE细胞后脂联素蛋白的表达。结果:①重组质粒AAV-MCS-Ad经双酶切和测序鉴定证实将脂联素基因已正确插入。②SDS-PAGE和电镜均证实rAAV-Ad病毒已构建成功。③Southernblotting测定病毒滴度约为约2.5×1014L-1,通过rAAV-LacZ转染细胞后X-gal染色计数,病毒的感染滴度在(0.83～1.2)×1010转导单位/mL。④Westernblotting证实rAAV-Ad转染H4IIE细胞后在30ku处见特异的阳性蛋白条带,证明能有效表达脂联素蛋白。结论:成功构建了携带小鼠脂联素基因的rAAV-Ad载体,经纯化浓缩后具有较好的纯度和较高滴度,能有效转染H4IIE细胞,并表达脂联素蛋白,具有良好的功能,为进一步研究其在真核细胞中的功能奠定了基础。     

小鼠脂联素重组腺相关病毒载体的构建

目的：构建脂联素重组腺相关病毒载体，为进一步研究脂联素的功能提供基础。方法：实验于2003年在中山大学附属第二医院林百欣医学研究中心进行。以带有小鼠脂联素基因的质粒peDNA3．0-Ad为模板，聚合酶链反应克隆脂联素基因，并将聚合酶链反应扩增产物定向克隆于pAAV-MCS载体，重组AAV-MCS-Ad质粒经双酶切和测序鉴定。随后采用磷酸钙转染法将pAAV-MCS-Ad、pAAV-RC，pAAV-Helper质粒共转染AAV-293细胞，包装得到重组腺相关病毒（rAAV-Ad）。回收病毒原液，采用PEG8000和氯仿纯化浓缩病毒。SDS-PAGE及透射电镜鉴定纯化的病毒，并采用Southern blot测定重组腺相关病毒的滴度。Western blotting检测rAAV-Ad转染H4IIE细胞后脂联素蛋白的表达。 结果：①重组质粒AAV-MCS-Ad经双酶切和测序鉴定证实将脂联素基因已正确插入。②SDS-PAGE和电镜均证实rAAV-Ad病毒已构建成功。③Southern blotting测定病毒滴度约为约2．5&;#215;10^14L^-1，通过rAAV-LacZ转染细胞后X-gal染色计数，病毒的感染滴度在（0．83～1．2）&;#215;10^10转导单位／mL。（少Western blotting证实rAAV-Ad转染H4IIE细胞后在30ku处见特异的阳性蛋白条带，证明能有效表达脂联素蛋白。 结论：成功构建了携带小鼠脂联素基因的rAAV-Ad载体，经纯化浓缩后具有较好的纯度和较高滴度，能有效转染H4IIE细胞，并表达脂联素蛋白，具有良好的功能，为进一步研究其茁真核细胞中的功能奠定了基础。     

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.     

Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus.

Recombinant adeno-associated virus (rAAV) vectors are under consideration for a wide variety of gene therapy applications. One of the limitations of the rAAV vector system has been the difficulty in producing the vector in sufficient quantity for adequate preclinical and clinical evaluation. A common method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not be feasible for clinical scale manufacturing, we have sought approaches for rAAV vector production that avoid transient transfection procedures. In previously reported work, we generated an AAV packaging cell line that produces infectious rAAV when the vector genome is transfected into the cell line as plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the E1 region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produced when the packaging cell line is infected with the hybrid adenovirus and wild-type adenovirus. This method avoids the need for plasmid transfection and is adaptable to large-scale manufacturing processes.     

携带半乳糖苷酶报告基因重组腺相关病毒体内转染骨组织的实验观察

目的:腺相关病毒载体因具有安全性好、免疫源性低、能感染分裂和非分裂细胞且能介导外源基因稳定长期表达等优点而备受瞩目,是最有希望应用于临床的病毒类载体。观察携带半乳糖苷酶报告基因的重组腺相关病毒(rAAV-LacZ)体内转染骨组织的效果。方法:实验于2005-01/2006-01在北京大学医学部第三医院和皖南医学院弋矶山医院骨科实验室完成。实验材料:8周龄雄性SD大鼠8只,体质量220g左右。rAAV-LacZ载体(本元正阳公司,病毒滴度为6×1012v·g/mL);Ⅰ型胶原海绵载体(上海其胜公司)。实验分组:将大鼠随机分为实验组和对照组,每组4只。实验方法:实验组4只大鼠在胫骨前外侧做骨缺损槽后,将滴有6×1011v·g病毒量的rAAV-LacZ和Ⅰ型胶原载体复合物植入骨缺损槽;对照组4只大鼠仅在胫骨骨缺损槽内植入Ⅰ型胶原载体。实验评估:①手术后6周取材行x-gal染色后,分别行标本大体病理观察和组织学观察以了解β-半乳糖苷酶特异表达的情况。②同时提取实验部位以远器官肝和心脏组织RNA,反转录-聚合酶链反应检测这些部位有无β-半乳糖苷酶特异表达。结果:8只SD大鼠均进入结果分析。①大体病理观察结果:实验组动物胫骨骨缺损处取材经x-gal染色后可见骨质蓝染,即有特异性β-半乳糖苷酶表达,而骨缺损临近处肌肉也表达β-半乳糖苷酶,Ⅰ型胶原载体植入部位以远的骨质和肌肉则没有特异性β-半乳糖苷酶的表达。②组织学观察结果:实验组骨缺损处肌肉肌纤维特异性表达β-半乳糖苷酶且没有明显的淋巴细胞局部浸润。蓝染的骨皮质切片显示骨陷窝内的骨细胞特异性表达β-半乳糖苷酶。对照组则没有实验组上述特异性β-半乳糖苷酶的表达。③反转录-聚合酶链反应结果:实验组实验部位有特异性β-半乳糖苷酶的表达,而其以远的心、肝器官内没有特异性β-半乳糖苷酶的表达。而对照组实验部位和实验部位以远的心、肝器官内均没有特异性β-半乳糖苷酶的表达。结论:腺相关病毒载体直接注射到骨缺损和骨折部位可以有效转染骨和临近肌肉组织,并能长期(6周)表达所携带的外源基因,具有一定的安全性。     

Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors.

Gene transfer into the skin is a promising approach to treat inherited or acquired dermatological diseases and systemic monogenic deficiencies. For this purpose, the efficient and sustained gene delivery into keratinocytes is of critical importance. Recombinant adeno-associated virus (rAAV) vectors hold the potential to achieve a long-term gene transfer into various human organs. In order to evaluate this potential for skin gene therapy, human keratinocytes were transduced in vitro with rAAV vectors encoding the reporter genes beta-galactosidase (rAAV/LacZ) or green fluorescent protein (rAAV/GFP). Using rAAV/LacZ at a multiplicity of infection (MOI) of five transducing particles per cell, up to 70% of human keratinocytes were transduced within 48 h. This effect was independent of individual skin donors and different body areas serving as the source for keratinocyte isolation. rAAV had no significant influence on cell viability, but induced a growth arrest in transduced keratinocytes. This growth arrest was overcome by replating cells in fresh media. rAAV/GFP-transduced keratinocytes could be passaged several times, expressed GFP for up to 50 days, and passed the transgene to their daughter cells, suggesting that keratinocyto precursor cells were also transduced. Taken together, the results suggest that rAAV is a promising gene transfer vehicle for skin gene therapy.     

Additional transduction events after subretinal readministration of recombinant adeno-associated virus

Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.     

Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector.

Fanconi anemia (FA) is a recessive inherited disease characterized by defective DNA repair. FA cells are hypersensitive to DNA cross-linking agents that cause chromosomal instability and cell death. FA is manifested clinically by progressive pancytopenia, variable physical anomalies, and predisposition to malignancy. Four complementation groups have been identified, termed A, B, C, and D. The gene for the FA complementation group C, FACC, has been cloned. Expression of the FACC cDNA corrects the phenotypic defect of FA(C) cells, resulting in normalized cell growth in the presence of DNA cross-linking agents such as mitomycin C (MMC). Gene transfer of the FACC gene should provide a survival advantage to transduced hematopoietic cells, suggesting that FA might be an ideal candidate for gene therapy. We demonstrated efficient transduction, expression, and phenotypic correction in lymphoblastoid cell lines derived from FA (C) patients using a recombinant adeno-associated virus (rAAV) vector containing the FACC gene. Molecular characterization of the transduced FACC gene showed an intact unrearranged proviral genome with expression sufficient to normalize cell growth, cell cycle kinetics and chromosomal breakage in the presence of MMC. These observations were extended by testing rAAV transduction in hematopoietic progenitor cells. Peripheral blood CD34+ cells isolated from a FA (C) patient and transduced with rAAV/FACC virus yielded 5-10-fold more progenitor colonies than mock-infected cells, consistent with genetic "rescue" of corrected cells. This is the first demonstration of rAAV gene correction in primary human hematopoietic progenitor cells and has important implications for gene therapy of hematopoietic disorders, specifically FA.     

Distribution of IgG subclasses after anti-hepatitis B virus immunization with a recombinant vaccine

To assess whether a different IgG subclass distribution was elicited in “low” and “high responders” after vaccination with recombinant hepatitis B virus surface antigen, we selected from 360 vaccine recipients 30 “lowresponder” subjects, with anti-HBs levels of 10–160 mIU/ml, and 40 “high-responder” subjects, with anti-HBs levels greater than 10,000 mIU/ml. In both groups all IgG subclasses were elicited in the anti-HBs response and the greatest contribution was that of IgG 1, followed by IgG2. IgG l was significantly less represented after the second (58%) and third doses (61%) of vaccine in “low responders” compared with “high responders” (65% and 69%). The relative percentage of IgG2 was significantly higher after the second (33%) and third (30%) doses of vaccine in “low responders” than in “high responders” (29% and 26%). In “low responders” the age of vaccine recipients significantly influenced the anti-HBs IgG subclass distribution: IgG2 and IgG4 production was positively correlated with age, whereas the opposite was observed for IgGl. These data support the evidence that: (1) IgGl and IgG2 subclasses are mainly involved in the specific anti-HBs response both in “high” and “low responders”; (2) the relative contribution of specific IgG2 to vaccination is higher in low responders and progressively increases with age.     

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.     

Custom adeno-associated virus capsids: the next generation of recombinant vectors with novel tropism

Recombinant gene delivery vehicles based on adeno-associated virus (rAAV) have emerged as promising vectors for the correction of genetic and acquired human disease states. These vectors possess many characteristics, including low pathogenicity and immunogenicity, and long-term gene expression after a single administered dose, that make them leading candidates for clinical gene therapy applications. Yet, the broad tissue tropism of the available AAV serotypes remains a disadvantage for the safest, most effective in vivo delivery of transgenes to target tissues. In addition, clinically relevant cell types exist that are poorly transduced by current rAAV vectors. As a result, increased efforts are now being made to tailor the tropism of rAAV to improve their transduction and selectivity profiles. Flexible, diverse methodologies have emerged that allow more control over the cell surface receptors rAAV employs for cell entry. These novel rAAV production strategies have resulted in unique vectors characterized by unique capsid protein sequences that employ alternative receptors, and have provided a better understanding of many basic aspects of the rAAV life cycle. This review aims to summarize the genetic methods currently being employed to customize rAAV capsids.     

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a genetic deficiency of beta-glucuronidase (GUS). We used a recombinant adeno-associated virus vector (AAV-GUS) to deliver GUS cDNA to MPS VII mice. The route of vector administration had a dramatic effect on the extent and distribution of GUS activity. Intramuscular injection of AAV-GUS resulted in high, localized production of GUS, while intravenous administration produced low GUS activity in several tissues. This latter treatment of MPS VII mice reduced glycosaminoglycan levels in the liver to normal and reduced storage granules dramatically. We show that a single administration of AAV-GUS can provide sustained expression of GUS in a variety of cell types and is sufficient to reverse the disease phenotype at least in the liver.     

High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap.

Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.