Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.     

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.     

Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.     

Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.     

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.     

Chromosomal integration pattern of a helper-dependent minimal adenovirus vector with a selectable marker inserted into a 27.4-kilobase genomic stuffer

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a neo(r) gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable neo(r) transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes.     

Design and packaging of adeno-associated virus gene targeting vectors

Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy.     

Large-scale analysis of adeno-associated virus vector integration sites in normal human cells

The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.     

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.     

AAV-mediated gene targeting methods for human cells

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10(-5) to 10(-2) per infected cell. These targeting frequencies are 1-4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ～10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.     

Targeted integration of DNA using mutant lox sites in embryonic stem cells.

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albertet al. (1995)Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox Por LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.     

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.     

Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.     

High-Fidelity Correction of Mutations at Multiple Chromosomal Positions by Adeno-Associated Virus Vectors

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325-330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.     

靶向肿瘤治疗的腺相关病毒载体

靶向性是肿瘤治疗取得成功的关键因素。病毒载体用于治疗肿瘤的过程中必须要求特异性作用于肿瘤细胞的同时降低对正常细胞的毒性。腺相关病毒(adeno-associated virus,AAV)较其他病毒载体具有免疫原性小、宿主范围广和介导基因可长期表达等优点,因此得到了广泛的应用。然而,AAV载体针对肿瘤的靶向性一直是近年研究的热点和难点。现就AAV载体治疗肿瘤的概况和靶向策略以及其安全性等方面作一综述。     

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.     

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.     

新型重组AAV5/5载体及包装系统

本研究组建了一种可用于规模化生产的以重组单纯疱疹病毒为辅助病毒的AAV5/5载体包装系统。首先,将5型腺相关病毒 (AAV5) 的rep和cap基因插入I型单纯疱疹病毒 (HSV-1) 基因组非必需基因UL2中,获得重组病毒rHSV1-rep5cap5。其次,构建一种携带AAV5 ITR的通用型载体质粒pAAV5neo,将报告基因EGFP插入pAAV5neo中,得到pAAV5neo-EGFP质粒。将pAAV5neo-EGFP质粒导入BHK-21细胞,用G418选择培养,挑选出表达EGFP并在重组病毒rHSV1-rep5cap5感染下能高效产生rAAV5/5-EGFP的单克隆载体细胞株C020。用rHSV1-rep5cap5感染C020细胞制备rAAV5/5-EGFP,用“氯仿处理-聚乙二醇/氯化钠-氯仿抽提”方法粗纯化rAAV5/5-EGFP。用100 kDa分子量截流超滤方法进一步纯化和浓缩,获得高纯度的rAAV5-EGFP。SDS-PAGE电泳分析可见3条特征性外壳蛋白带。电镜分析显示病毒颗粒以实心颗粒为主。用rAAV5/5-EGFP病毒按1×105 vg/cell感染体外培养的HEK293细胞,可见30％细胞呈现绿色荧光。本研究提出了一种高效AAV5/5载体生产系统和纯化方法,为重组AAV5载体的进一步应用提供了基础。