Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication

In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.     

Adeno-associated virus vectors.

Adeno-associated virus is a human parvovirus that integrates its DNA genome into host cell chromosomes with very high efficiency. This suggests that adeno-associated virus may be a useful vector for human gene therapy. Interest in adeno-associated virus vectors increased greatly in the last year following reports that adeno-associated virus genome integration may be site specific and occur at preferred sites in the human genome. Several genes relevant to the treatment of genetic or infectious diseases have been expressed in adeno-associated virus vectors in vitro.     

Tamplicon-7, a Novel T-Lymphotropic Vector Derived from Human Herpesvirus 7

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

Novel strategy for generation and titration of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.     

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.     

Molecular cloning of the avian myelocytomatosis virus genome and recovery of infectious virus by transfection of chicken cells.

The avian retrovirus myelocytomatosis virus 19 (MCV) possesses an interesting diversity of oncogenic potentials, but the virus has proven difficult to study because of its inability to replicate without the assistance of a helper virus. We have therefore isolated and amplified the genome of MCV by molecular cloning in a procaryotic vector. The topography of the cloned DNA was explored by the use of restriction endonucleases and radioactive complementary DNAs representing specific domains in avian retrovirus genomes. The cloned DNA appeared to be an authentic representation of the MCV genome: the size and genetic topography of the DNA were comparable to those of MCV, and transfection of the cloned DNA into chicken cells (in company with the DNA of a suitable helper virus) gave rise to virus with the genome and transforming potentials of MCV. The availability of cloned MCV DNA should facilitate a variety of genetic and biochemical manipulations directed at elucidating the mechanism of oncogenesis by MCV.     

A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy

Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.     

重组禽腺联病毒介导的输卵管暂态生物反应器的初步研究

将输卵管特异表达启动子调控的人组织激肽释放酶（human tissue kallikrein,hKLK1）表达盒插入至AAAV转移载体pAITR中,与AAAV包装载体pcDNA-ARC及腺病毒辅助质粒pHelper三质粒利用磷酸钙沉淀法共转染AAV-293细胞,制备输卵管特异表达hKLK1的rAAAV。将获得的重组病毒以每只鸡2×1010病毒颗粒数翅静脉注射正常产蛋母鸡,RT-PCR结果显示hKLK1只在输卵管部位表达;酶活性检测结果表明：注射后第2天就可以检测到rhKLK1的活性,第3周表达量最高,达107.3U/ml,表达时间持续6周之久;用含rhKLK1的蛋清灌喂自发性高血压大鼠（SHR）,可使其血压下降70mmHg,5天后回升到饲喂前水平。以上结果表明重组禽腺联病毒介导的鸡输卵管暂态生物反应器不仅具有很好的组织特异性,而且可指导外源基因长期稳定的表达。     

Replication of adeno-associated virus in synchronized cells without the addition of a helper virus.

We investigated the helper-independent replication of adeno-associated virus (AAV) in cells synchronized by pretreatment with hydroxyurea, reversal of polyamine depletion, or physical mitotic detachment. In Chinese hamster cells (OD4 line) treated with hydroxyurea prior to infection. AAV underwent a complete cycle of replication. Transfection of such cells with plasmid-cloned AAV DNAs also gave rise to infectious viral progeny. Synchronization of OD4 cells by reversal of polyamine depletion or mitotic detachment led to independent AAV DNA synthesis (and infectious viral progeny in the case of the former procedure), but these procedures were not as effective as hydroxyurea pretreatment. Independent AAV DNA synthesis was also detected in some other cell lines of Chinese hamster, human, and monkey origin treated with hydroxyurea prior to infection. The results demonstrate that, in contrast to previous notions, the AAV infectious process is not absolutely dependent upon the addition of a coinfecting helper virus.     

Chimeric retroviral helper virus and picornavirus IRES sequence to eliminate DNA methylation for improved retroviral packaging cells

Most retroviral packaging cell lines were established by a helper virus plasmid cotransfected with a separate plasmid encoding a selection marker. Since this selection marker coexisted in trans with the helper virus sequence, helper virus gene expression could be inactivated by host DNA methylation despite selection for the cotransfected selection marker. We have reported that DNA methylation could occur in the long terminal repeat (LTR) region of helper virus in vector producer cells (VPC) in up to 2% of the population per day (W. B. Young, G. L. Lindberg, and C. J. Link, Jr., J. Virol. 74:3177-3187, 2000). To overcome host cell DNA methylation that suppresses viral gene expression, we constructed a chimeric retroviral helper virus, pAM3-IRES-Zeo, that contains Moloney murine leukemia virus as a helper virus and a picornavirus internal ribosome entry site (IRES) sequence followed by a Zeocin selection marker at the 3' end of the env sequence. This pAM3-IRES-Zeo permitted selection for intact and functional helper virus in transfected cells without subcloning. By selection with Zeocin, a mixed population of pAM3-IRES-Zeo-transfected NIH3T3 cells (AMIZ cells) was maintained with little or no DNA methylation of the helper virus 5' LTR. The high level of pAM3-IRES-Zeo gene expression resulted in no detectable vector superinfection and in high vector titers (2 x 10(6) to 1.5 x 10(7) CFU/ml) after introduction of a retroviral vector. When Zeocin selection was withdrawn from AMIZ cells, methylation of the 5' LTR increased from 17 to 36% of the population during 67 days of continuous culture and the cells became susceptible to superinfection. During this period, gene expression of pAM3-IRES-Zeo decreased and vector titer production was reduced to 2 x 10(4) CFU/ml. These data demonstrate an important role of DNA methylation in the genetic instability of VPC. The chimeric helper virus allows the establishment of a mixed population of packaging cells capable of high-level and sustained vector production without cloning procedures.     

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.     

The evolution of defective and autonomous parvoviruses.

Because of the small size and genetic simplicity of small DNA viruses, parvoviruses would appear to be excellent models for studying viral evolution and adaptation. In an earlier publication we hypothesized the evolution of sequences of cellular "junk" DNA into protective interfering transposons. These transposons would interfere with invading pathogenic viruses by competing with the pathogen DNA for replicative enzymes. We speculated that a small, defective parvovirus, the adeno-associated virus (AAV), which usually requires the presence of a pathogenic helper virus to replicate, may have evolved from such a piece of cellular "junk" DNA. Our theory predicted that AAVs, as a consequence of their defective nature, developed under pressures favoring maintenance of their transposon like qualities. In contrast, disease-causing, autonomous, non-defective parvoviruses such as the B19 agent of humans and the canine parvovirus, even though their origins may have been in cellular DNA, would appear to have developed under totally different evolutionary pressures. In this paper we will present evidence for a common ancestry for the defective and autonomous parvoviruses and discuss the divergent paths this evolution may have taken in establishing the two genera.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains

The subnuclear distribution of replication complex proteins is being recognized as an important factor for the control of DNA replication. Herpes simplex virus (HSV) single-strand (ss)DNA-binding protein, ICP8 (infected cell protein 8) accumulates in nuclear replication domains. ICP8 also serves as helper function for the replication of adeno-associated virus (AAV). Using quantitative 3D colocalization analysis we show that upon coinfection of AAV and HSV the AAV replication protein Rep and ICP8 co-reside in HSV replication domains. In contrast, Rep expressed by a recombinant HSV, in the absence of AAV DNA, displayed a nuclear distribution pattern distinct from that of ICP8. Colocal ization of Rep and ICP8 was restored by the reintroduction of single-stranded AAV vector genomes. In vitro, ICP8 displayed direct binding to Rep78. Single-stranded recombinant AAV DNA strongly stimulated this interaction, whereas double-stranded DNA was ineffective. Our findings suggest that ICP8 by its strong ssDNA-binding activity exploits the unique single-strandedness of the AAV genome to form a tripartite complex with Rep78 and AAV ssDNA. This novel mechanism for recruiting components of a functional replication complex directs AAV to subnuclear HSV replication compartments where the HSV replication complex can replicate the AAV genome.     

A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication.

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.