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.     

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.     

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.     

Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors

Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.     

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.     

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.     

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.     

Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.     

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.     

Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells.

Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.     

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.     

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。     

An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.     

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.     

Latent infection of KB cells with adeno-associated virus type 2.

Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV-specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular-weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo.     

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.     

Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site,AAVS1, on human chromosome 19

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity ( approximately 4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.     

Gene targeting using zinc finger nucleases

The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.