Adeno-associated virus vectors for gene transfer to the brain

Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinson's disease.     

Development and application of gene therapy technologies

The success of hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency (X-SCID) was a major breakthrough in the field of gene therapy. However, two patients treated with this gene therapy developed leukemia at a later time, and retroviral vector-mediated gene transfer was considered to trigger leukemogenesis; i.e. insertional mutagenesis caused activation of LMO 2 gene, which was one step toward leukemia development. To cope with this serious problem, basic studies are required to improve the safety of retroviral vectors and to develop the method for site-specific integration of transgenes. In addition, we have to develop technologies such as selective amplifier genes (SAGs), the system for selective expansion of transduced cells, in order to obtain therapeutic efficacy of hematopoietic stem cell gene therapy in many other disorders. Moreover, clinical applications of AAV vector are promising from the standpoint of safety issue, because this vector is derived from non-pathogenic virus. AAV vector is appropriate for gene transfer into neurons, muscles, and hepatocytes. For example, gene therapy for Parkinson's disease is investigated using AAV vectors. Genetic manipulation is also one of the indispensable technologies in the field of regeneration medicine, and further promotion of basic research is important.     

利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一,产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒,建立双病毒感染昆虫细胞以生产AAV的技术体系。首先,进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增,并检测杆状病毒滴度及其感染细胞的效果;然后,使用双杆状病毒共感染昆虫细胞,并优化感染条件;最后,基于优化条件进行AAV生产,并检测评估产量、质量等相关指标。结果表明,AAV生产用多基因缺失型杆状病毒滴度较野生型无差异,感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产,Bac4.0-1的基因组滴度为1.63×10¹¹ VG/mL,Bac5.0-2的基因组滴度为1.02×10¹¹ VG/mL,较野生型产量分别提升了240%和110%。电镜下,3组均具有正常的AAV病毒形态,且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系,显著提高了AAV产量,具有一定的应用价值。     

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.     

Novel AAV serotypes for improved ocular gene transfer

Some of the most successful gene therapy results have been obtained using recombinant viral vectors to treat animal models of inherited and acquired ocular diseases. Clinical trials using adenovirus vector systems have been initiated for two ocular diseases. Adeno-associated viruses (AAVs) represent an attractive alternative to adenoviral vector systems as they enable stable and long-term expression and can target a variety of different ocular cell types depending on the capsid serotype; recently clinical trails for congenital blindness was initiated with a vector-based AAV serotype 2. High levels of retinal gene transfer have been achieved using vectors based on AAV serotypes 1, 2, 4 and 5. This report compares the gene transfer efficacy and stability of expression of vector systems based on three novel AAV serotypes: AAV7, 8, 9, with the established vectors AAV1, 2, 5. We show here that AAV7 and 8 enable superior long-term transduction of retinal and also anterior chamber structures.     

Structure of adeno-associated virus serotype 8, a gene therapy vector

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-A resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded beta-barrel and long loops between the beta-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.     

新型核心蛋白聚糖靶向性重组腺相关病毒的制备与鉴定

核心蛋白聚糖(decorin, DCN)是广泛存在于细胞基质中的一种富含亮氨酸的蛋白多糖, 属于蛋白聚糖家族中的小分子类. DCN可作为多种细胞因子的配体, 发挥多种生物学功能. DCN在一些肿瘤组织中高水平表达,调控恶性肿瘤的生长和迁移. 腺相关病毒(AAV)是肿瘤基因治疗中常用的基因工程载体, 利用重组技术可以实现对病毒衣壳蛋白的改造, 使其感染具有靶向性. 而针对DCN高表达细胞的转导可能成为肿瘤基因治疗应用中定向导入治疗基因的有效策略. 本研究在对多种DCN结合蛋白序列保守区的分析基础上, 筛选出具有较高活性的DCN结合功能域(DB1), 并将其融合至AAV衣壳蛋白VP2编码序列的N端; 继而利用AAV的嵌合包装技术, 成功制备了衣壳展示DB1表位的重组AAV. 在过表达DCN细胞的感染实验中, 该病毒表现出针对DCN较强的靶向性. 本研究所制备的DCN靶向性腺相关病毒不仅为肿瘤治疗的应用提供了一种新型载体, 同时可作为一类特殊的基因导入工具为研究DCN在肿瘤发生发展中的作用提供帮助.     

Gene therapy using AAV

AAV (adeno-associated virus) vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells. Among various neurological disorders, Parkinson's disease (PD) is one of the most promising candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. One of the major approaches to gene therapy of PD is the intrastriatal expression of dopamine (DA)-synthesizing enzyme genes. As for the initial step of clinical application, AAV vector-mediated AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by adjusting the dose of L-DOPA. Second, intramuscular injection of AAV vectors is appropriate to protein-supplement gene therapy. Monogenic diseases such as hemophilia and Fabry disease are suitable candidates. Regarding cancer gene therapy, AAV vectors may be utilized to inhibit tumor angiogenesis, metastasis, and invasion. When long-term transgene expression in stem cells is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of AAV would be particularly valuable.     

Establishment of an AAV reverse infection-based array

Background

The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited.

Principal Findings

We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments.

Conclusions/Significance

Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays.     

Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.     

Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene

The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.     

Separate control of rep and cap expression using mutant and wild-type LoxP sequences and improved packaging system for adeno-associated virus vector production

Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing. To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors. In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system. Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences. The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre. By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase. When the LacZencoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 × 10¹⁰ genome copies per 10-cm dish was obtained. These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.     

Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.     

Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5-rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.     

The Use of Heterologous Promoters for Adeno-Associated Virus (AAV) Protein Expression in AAV Vector Production

Although adeno-associated virus (AAV) vectors are potentially useful gene transfer vehicles for gene therapy, the vector production system is currently at the developmental stage. We constructed AAV helper plasmids (Rep and Cap expression plasmids) by replacing a native AAV promoter, p5, with various heterologous promoters to examine whether the efficiency of AAV vector production was influenced by modulating the AAV protein expression pattern. The helper plasmids containing heterologous promoters (EF, CMV, SV40, B19p6, and CAG promoters, respectively) expressed Rep78/68 more efficiently than a conventional helper plasmid (pIM45), but the expression of Rep52/40 and Cap decreased, resulting in a significant reduction in AAV vector production. Furthermore, the efficiency of vector production never fully recovered even if the Cap proteins were supplied by an additional expression plasmid. A large amount of Rep78/68 and/or a reduced level of Rep52/40 may have deleterious effects on AAV vector production. The present findings will aid in the development of a more efficient AAV vector production system.     

Construction and expression of a recombinant adeno-associated virus that harbors a human beta-globin-encoding cDNA.

Towards a goal of using recombinant adeno-associated viruses (AAV) for the gene therapy of hemoglobinopathies we had previously constructed plasmid pAV h beta G psi 1, which contained a human beta-globin-encoding cDNA (HBB) downstream from the P40 promoter of AAV2 DNA [Ohi et al., Gene 89 (1990) 279-282]. Transfection of the plasmid into human 293 cells (embryonal kidney cell line) resulted in the expression of HBB at the mRNA level as well as rescue and replication of the recombinant AAV genome (Ohi et al., ibid.). The present study demonstrates that the replicated recombinant DNA was packaged into an intact virion by transcomplementation with pAV2 or the defective helpers, pAV delta Bam or pAVXB. The recombinant virus could be isolated by equilibrium CsCl density gradient, the density of which was about 1.4 g/cm3. The defective helpers are used to produce wild-type AAV-free recombinant AAV. The recombinant AAV were infectious and expressed chimeric mRNAs containing the HBB sequence in virus-infected 293, KB (oral epidermoid carcinoma cell line) and K562 (human erythroleukemia cell line) cells. The importance of the infectivity and expression of the recombinant AAV in hematopoietic cells is discussed in the context of gene therapy of hemoglobinopathies.     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.     

2型腺相关病毒转导人胎肝造血细胞及其介导的β珠蛋白基因的表达

β地中海贫血是一种因β珠蛋白基因缺陷导致的遗传性贫血性疾病,基因治疗是唯一有望治愈该病的方法.2型腺相关病毒(adeno-associated virustype2,AAV2)是一种非致病性病毒,作为一种基因治疗载体,其应用潜力日益受到关注.目前还未见AAV2转导人早期胎肝造血细胞及其介导β珠蛋白基因在动物体内表达的实验报道.有研究表明,AAV2转导人造血干细胞的效率,因各实验室包装和纯化rAAV2的方法不同而存在差异,其中辅助病毒的污染被认为是一重要原因.制备了无辅助病毒污染的rAAV2,经体外检测其滴度,纯度及功能后,再转导人早期胎肝造血细胞,将被转导的胎肝造血细胞移植入受亚致死量剂量照射的8只BALB/C裸鼠体内,检测rAAV2介导的β珠蛋白基因在裸鼠体内的表达.结果显示:制备的无辅助病毒污染的rAAV2具有较高的滴度、纯度,并能够在体外介导β基因的表达;在8只受试BALB/C裸鼠中,RT-PCR在2只BALB/C裸鼠骨髓中检测到β珠蛋白基因的表达.提示,rAAV2能够转导人早期胎肝细胞并介导β珠蛋白基因的表达,但同时也存在表达量较低的缺点,应用于β地中海贫血的基因治疗还需要对AAV2生物学特性做深入的研究.     

利用BmNPV-家蚕表达系统包装重组腺相关病毒2型（rAAV2）研究

腺相关病毒（adeno-associated virus,AAV）是基因治疗中最常用的病毒载体之一,目前用于基因治疗的AAV多利用苜蓿银纹夜蛾核型多角体病毒表达系统（AcMNPV-sf9）包装,但较高的包装成本限制了AAV在基因治疗中的广泛应用。家蚕杆状病毒表达系统与AcMNPV-sf9系统相比,具有包装量更高、成本更低的优势,因此更适用于包装重组腺相关病毒（recombinant adeno-associated virus,rAAV）。首先,将AAV2功能基因cap和rep进行序列优化后合成,克隆到杆状病毒转移载体pVL1393上,将增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因和荧光素酶（luciferase,Luc）基因分别作为报告基因克隆到含有巨噬细胞病毒IE（cytomegalovirus-IE,CMV-IE）启动子的病毒转移载体pVL1393-ITRs-MCS上。随后,将构建好的转移载体分别与缺陷型家蚕杆状病毒reBmBac共转染BmN细胞系,获得分别重组有cap、rep和报告基因的家蚕杆状病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）。再将纯化的重组病毒（reBm-Cap2、reBm-Rep2）与reBm-EGFP、reBm-Luc分别混合后感染家蚕,收获其表达产物,纯化得到含有目的基因的rAAV病毒。利用rAAV病毒感染哺乳动物细胞后,通过检测EGFP、Luc的表达状态来验证rAAV包装成功与否。结果显示,利用家蚕杆状病毒系统成功包装了rAAV2,并且在哺乳动物细胞中实现了报告基因的表达。