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.     

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.     

Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.     

Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.     

Recent developments in adeno-associated virus vector technology

Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges.     

Role for highly regulated rep gene expression in adeno-associated virus vector production.

Recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (AAV) vectors (X. Xiao, J. Li, and R. J. Samulski, J. Virol. 70:8098-8108, 1996) has encouraged further development of this vector for human gene therapy. Currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (Ad)-infected cells. To examine the effects of the AAV replication (rep) genes on recombinant AAV (rAAV) vector production, we have constructed a series of AAV helper plasmids that contain strong heterologous promoters in place of the endogenous p5 promoter. Although high-level rep gene expression was achieved, rAAV DNA failed to replicate in the absence of Ad infection. Moreover, unregulated overexpression of Rep78/68 led to substantially lower rAAV yields in the presence of Ad (10(4-5) versus 10(7-8)). In contrast, under similar conditions, reduced Rep78/68 expression resulted in much higher rAAV yields (10(9)). Molecular characterization showed that overexpression of the rep gene decreased rAAV DNA replication and severely inhibited capsid (cap) gene expression. Interestingly, a reduced rep level enhanced cap gene expression and supported normal rAAV DNA replication. These studies suggest a critical role for regulated rep gene expression in rAAV production and have facilitated the development of a new AAV helper plasmid that increases vector production eightfold over currently used constructs.     

Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product.

We previously described use of the human parvovirus, adeno-associated virus (AAV), as a vector for transient expression in mammalian cells of the gene for chloramphenicol acetyltransferase (CAT). In the AAV vector, pTS1, the CAT gene is expressed under the control of the major AAV promoter p40. This promoter is embedded within the carboxyl-terminal region of an open reading frame (orf-1) which codes for a protein (rep) required for AAV DNA replication. We show here that the rep product has additional trans-acting properties to regulate gene expression. First, deletion or frame-shift mutations in orf-1, which occurred far upstream of p40, increased expression of CAT in human 293 (adenovirus-transformed) cells. This increased CAT expression was abolished when such mutant AAV vectors were transfected into 293 cells together with a second AAV vector which could supply the wild-type AAV rep product in trans. Thus, an AAV rep gene product was a negative regulator, in trans, of expression of CAT in uninfected 293 cells. In adenovirus-infected 293 cells, the function of the AAV rep product was more complex, but in some cases, it appeared to be a trans activator of the expression from p40. In HeLa cells, only trans activation by rep was seen in the absence or presence of adenovirus. Neither activation nor repression by the rep product required replication per se of the AAV vector DNA. Thus, trans-acting negative or positive regulation of gene expression by the AAV rep gene is modulated by factors in the host cell and by the helper adenovirus.     

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.     

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.     

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.     

Gene therapy with the TRF1 telomere gene rescues decreased TRF1 levels with aging and prolongs mouse health span

The shelterin complex protects telomeres by preventing them from being degraded and recognized as double‐strand DNA breaks. TRF1 is an essential component of shelterin, with important roles in telomere protection and telomere replication. We previously showed that TRF1 deficiency in the context of different mouse tissues leads to loss of tissue homeostasis owing to impaired stem cell function. Here, we show that TRF1 levels decrease during organismal aging both in mice and in humans. We further show that increasing TRF1 expression in both adult (1‐year‐old) and old (2‐year‐old) mice using gene therapy can delay age‐associated pathologies. To this end, we used the nonintegrative adeno‐associated serotype 9 vector (AAV9), which transduces the majority of mouse tissues allowing for moderate and transient TRF1 overexpression. AAV9‐TRF1 gene therapy significantly prevented age‐related decline in neuromuscular function, glucose tolerance, cognitive function, maintenance of subcutaneous fat, and chronic anemia. Interestingly, although AAV9‐TRF1 treatment did not significantly affect median telomere length, we found a lower abundance of short telomeres and of telomere‐associated DNA damage in some tissues. Together, these findings suggest that rescuing naturally decreased TRF1 levels during mouse aging using AAV9‐TRF1 gene therapy results in an improved mouse health span.     

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.     

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3′ end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.     

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.     

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.     

Intramuscular Injection of AAV8 in Mice and Macaques Is Associated with Substantial Hepatic Targeting and Transgene Expression

Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.     

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.     

Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.     

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.