Adeno-associated virus (AAV) vectors for gene transfer

Advanced Drug Delivery Reviews, 12 (1993) 201-215

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{) 1993 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/93/$24.00

A D R 00168

Adeno-associated virus (AAV) vectors for gene transfer Xiao Xiao, Wanda deVlaminck and John Monahan Avigen, Inc., Alameda, CA, USA (Received A u g u s t 6, 1993) (Accepted A u g u s t 16, 1993)

Key words: G e n e therapy; Integration; N o n - p a t h o g e n i c

Contents Summary .................................................................................................................

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I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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II. General description o f adeno-associated virus ( A A V ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. G e n o m e structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Rep proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. C a p proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Inverted terminal repeat (ITR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I11. Life cycle o f a d e n o - a s s o c i a t e d virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Regulation o f A A V gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Specific integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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IV. A d e n o - a s s o c i a t e d virus as a vector for gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. P a c k a g i n g system free o f wild-type A A V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. H o s t range o f A A V vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Expression o f t r a n s d u c e d genes in A A V vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V. A d v a n t a g e s a n d limitations of A A V vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VI. C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements .....................................................................................................

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Correspondence to: John Monahan, Ph.D., Avigen, Inc., 1201 Harbor Bay Parkway #1000, Alameda, CA 94540, USA.

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Summary One of the most promising virus vectors being studied for gene therapy is derived from adeno-associated virus (AAV), a single-stranded D N A virus that is ubiquitous to humans but has never been associated with any disease. AAV has characteristics that make it an obvious candidate for development as a gene therapy vector. This virus infects dividing or quiescent cells and exhibits stable, non-random integration into the genome of the target cells. In addition, AAV has a broad host and tissue range. Viral particles are extremely stable which is a significant advantage for scaleup and commercialization. A great deal of progress has been made in the last 10 years towards understanding the genetics and molecular biology of AAV). Gene transfer studies have been done by a number of investigators who have shown that AAV vectors can be used to transduce a variety of cells at high efficiencies. The major challenges for the future are to develop packaging cell lines for large-scale production of AAV vectors and to develop animal models for clinical studies.

I. Introduction Gene therapy is a new and rapidly evolving technology that potentially can treat a host of genetic and acquired diseases by using a patient's own genetically modified cells to produce desired gene products such as proteins and R N A molecules. Genetic modification of cells has been accomplished through a number of physical and chemical methods; however, virus vectors have proven to be the most effective method for gene delivery. A wide variety of virus vectors have been developed for research purposes based on murine retroviruses, adenoviruses, herpes viruses and pox viruses. To date, however, the only virus vectors used for clinical studies are derived from murine retroviruses and adenoviruses. AAV-derived vectors are emerging as possible candidates for use in gene therapy because they have a number of potential advantages over vectors derived from other viruses [2-4]. AAV is non-pathogenic and has never been associated with disease even though more than 85% of the US population is seropositive for the virus [1]. AAV integrates stably into the genome of a variety of cells with high efficiency and in a non-random manner. Also, AAV is resistant to solvents and detergents as well as extremes of temperature and pH. The difficulties in studying the genetic features of AAV were overcome with Samulski's work in 1982 when he and his colleagues cloned the entire AAV genome into bacterial plasmids and demonstrated that those plasmids were infectious [5]. This work enabled studies to be done with different vector constructs and mutants and has contributed to our present understanding of the structure and function of AAV. These scientific advances and the unique features of AAV have provided the basis for developing AAV vectors for gene therapy and one can anticipate that AAV vectors will be used in clinical studies within the next several years.

II. General description of adeno-associated virus AAV is a member of the Parvoviridae family of small D N A animal viruses. In

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addition to one genus of insect viruses (densoviruses), this family of viruses consists of two genera that infect vertebrates ranging from birds to humans. Parvovirus, which includes a human pathogen B-19 virus and a canine parvovirus, are referred to as "autonomous viruses" because they can replicate in dividing cells without coinfection with helper virus. In contrast, AAV, a member of the dependovirus genus, requires helper virus such as adenovirus or herpesvirus for its productive growth [1,6]. Nevertheless, when the host cells are exposed to stressful conditions such as genotoxic treatment or synchronization, the viral D N A can replicate to a certain extent without the requirement of helper viruses. Currently more than six different AAV viruses have been isolated, including strains from humans, monkeys, birds and pigs [3,7]. AAV-2, isolated from human cells, has been studied most extensively and will be the focus of this review [8]. AAV is one of the smallest non-enveloped D N A viruses and localizes in the nucleus of the host cell. The virion is an icosahedron of about 20-22 nm in diameter and is comprised of 26% D N A and 74% protein with a density of 1.41 g/cm 3 [9-11]. The AAV particles are stable in a wide pH range between 3 and 9 and are resistant to heating at 56~C for least for 1 hour, to treatment with many detergents such as deoxycholate and organic solvents such as chloroform and ether. In a typical productive infection, a virus titer as high as 10m-10 t~ infectious particles/ml can be obtained. The virus also can be readily purified and concentrated by CsC1 gradient centrifugation and is stable when stored in solution or in the lyophilized form. These remarkable stability characteristics have significant implications for commercial development of AAV vectors.

11.1. Genome structure The AAV-2 genome is a linear, single-stranded D N A consisting of 4680 bases [12]. Both plus and minus strands of D N A are packaged with equal efficiency into individual viral particles and are equally infectious [13]. There are three important functional components in the AAV genome: two major open reading frames and the inverted terminal repeats (Fig. 1). The open reading frame in the left half of the genome contains the Rep (replication) gene and the one in the right half contains the Cap (capsid) gene. Three promoters have been identified and named according to their approximate map positions, p5, p19, and p40. Promoters p5 and p19 regulate the expression of Rep proteins and p40 controls the Cap proteins. Messenger RNA's initiated at the three different promoters all terminate at the same polyadenylation site at map position 96. At both ends of the genome reside the 145 base pair (bp) inverted terminal repeat (ITR), of which the first 125 nucleotides form a selfcomplementary "T"-shaped hairpin structure, consisting of a stem of 41 base pairs together with two arms of 9 bp.

11.2. Rep prolehz,s' Four Rep proteins have been identified. Rep78 and Rep68 are derived from promoter p5, while Rep52 and Rep40 are from promoter p19; Rep68 and Rep40 are the products from the spliced m R N A ' s [14,15]. All four Rep proteins share a

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common C-terminal region and differ only at the N-termini, depending upon promoter usage and splicing status (Fig. 1). Rep proteins, especially Rep78 and Rep68, are multifunctional proteins; however, their major function is for AAV-DNA replication [3,16]. Genetic and biochemical studies have demonstrated that the two larger proteins (Rep78 and Rep68) can bind to the AAV replication origin, i.e. the terminal repeat. They display a site-specific and strand-specific nickase, a helicase and ATPase activity, which are critical for AAV-DNA replication. The functions of Rep52 and Rep40 are difficult to characterize, because their coding region overlaps in-frame and resides within the two larger Rep proteins. The only clue to their function was obtained by a mutation at the A U G codon of the Rep52 and Rep40, which suggestes that these two proteins play some role in the viral ssDNA accumulation [17]. However, mutations of the two larger Rep proteins displayed almost all known rep phenotypes. Besides DNA replication, other functions also have been assigned to Rep proteins, including autoregulation of AAV gene expression, repression of heterologous promoters, inhibition of oncogene amplification, and inhibition of cell transformation by oncogenic viruses [for review, see Refs. 1, 3 and 16 and references therein[. For example, AAV gene products, Rep 78 and 68, can regulate all three AAV promoters, p5, p19 and p40 in either positive or negative manners, depending on the presence or absence of adenovirus [18,19].

11.3. Cap proteins The AAV viral particle consists of three capsid proteins, VP1, VP2 and VP3 with molecular weights of 87, 73 and 61 kD respectively. VP3 is the major component of the virion and represents 86% of the virion proteins. VP2 and VP3 account for about 6% each. All Cap proteins are derived from a single promoter p40. Like Rep proteins, the three Cap proteins share their identical C-terminal region and differ only at the N-termini. VP2 and VP3 are translated from the same 2.3 kb spliced mRNA, although VP2 initiates at an alternative ACG codon upstream of the A U G codon of VP3 [20]. VP1 is synthesized from a 2.3 kb alternatively spliced m R N A (Fig. 1) [21,22]. Mutations in the VPI region cause a phenotype named lip representing low yields of infectious particles [23,24]. Mutations in VP2 and VP3 regions eliminate virion formation and ssDNA accumulation.

11.4. Mverted terminal repeat (ITR) The special structural feature of AAV-ITRs plays a critical role in AAV-DNA replication, since the fold-back 3'-terminus can act as a primer for single-stranded viral DNA synthesis. Genetic and biochemical studies have demonstrated that the ITR is the origin of replication [1,3,16]. Recently, more evidence revealed that the 145 bp ITR is the only cis-element required for all the steps of the AAV life cycle, including rescue from the host genome, replication, packaging and integration, etc. [Xiao, X. and Samulski, R.J., unpublished results; Muzyczka, N., personal communication]. Although the ITRs have been considered transcriptionally silent in the absence of Rep proteins, a recent report suggested certain promoter functions of the ITRs [25].

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Fig. I. Map of the AAV-2 genome. The solid segments at the two ends of the AAV-2 genome (top) designate the inverted terminal repeats. The three arrows (p5, p19, p40) designate transcriptional start sites for AAV promoters.

III. Life cycle of adeno-asociated virus AAV can assume two life cycles upon infection into host cells: latent and lyric. For latent infection, AAV stably integrates into the genome of the host cell where it remains dormant until the cell becomes infected with a helper virus such as adenovirus or herpesvirus. The ability of the AAV genome to remain dormant but to be transmitted to progeny as a result of cell division is not only an effective strategy for virus survival but makes it an appealing candidate for use as a vector for the introduction of non-viral genes into cells. In the presence of helper virus, replication occurs and the AAV genome is excised and proceeds through a normal, productive infection.

IIl.1. Regulation of AA V gene expression In the lytic infection, regulation of AAV gene expression is rather complex. Besides its own regulatory mechanisms, it is dependent upon and regulated by helper viruses. In addition, AAV can also counter-regulate the helper viral gene expression [1,3,7]. During the early stage of adenovirus co-infection, E1A proteins from adenovirus, the earliest expressed helper function for AAV, greatly stimulates the p5 promoter and the E1A [26]. Consequently, the p5 products Rep78 and possibly Rep68 can further stimulate all three AAV promoters [18,19,27,28]. As the infection proceeds, transcripts from all three promoters gradually increase until they reach a steady state at approximately 10 hours post-infection. Therefore, unlike many other viruses, AAV gene expression does not have an obvious early to late switch. Although herpesvirus and vaccinia virus are able to provide helper functions to AAV, adenovirus is the most commonly utilized helper virus. Five gene products of adenovirus are required for a fully permissive AAV lytic infection. These gene products are E1A, E1B, E4, E2A and VA-RNA [1,3,7]. E1A is responsible at least for the AAV p5 promoter activation through the interaction with host transcription

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factor YY-1 [29]. E1B and E4 products are believed to stabilize and facilitate the transportation of AAV-RNAs [30-34]. E2A is an adenovirus ssDNA binding protein and does not have obvious effects on A A V - D N A replication. However, its presence seems necessary for efficient A A V - R N A splicing and translation [35,36]. The VA-RNAs of the adenovirus have been demonstrated to increase the steadystate level of AAV-RNAs, and also to stimulate their efficient translation, especially the p40 capsid m R N A [30, 3541]. During latent infection with wild-type AAV, the viral gene expression is generally repressed. It is believed that the Rep gene products are partly responsible for the repression. Although neither AAV transcripts nor the proteins are detectable, there exists some indirect evidence for low-level Rep gene expression during latent infection [18,19,27].

111.2. Replication Since AAV possesses two inverted terminal repeats (ITRs), it is conceivable that the virus replicates its D N A via the hairpin transfer model during the lytic infection [42, 43]. Upon entering the cells, the AAV virion particles de-coat in the nucleus and release their single-stranded D N A genome. The initial conversion of single-stranded D N A into duplex D N A occurs by using the 3'-end hairpin as a primer for the fill-in replication, to form a large T-shaped hairpin intermediate. As a result, the entire coding region as well as the ITR on the 5'-end is completely replicated. To replicate the 3'-end ITR, a nick is made by Rep proteins on the parental strand at the 3'-ITR region to generate a new internal 3'-hydroxyl group. This nick is then used as a primer to finish the replication of the 3'-ITR. At this point the entire genome is replicated. This duplex replication form (RF) D N A is subsequently amplified to a large pool. From this pool, a single-stranded AAV genome is packaged into the preformed empty capsid to produce mature virions [for more detail, see Refs. 3 and 16].

111.3. Specific integration Like retroviruses, AAV utilizes integration as a necessary step in its life cycle. AAV was initially discovered as a cryptic virus in many cell lines, both primary and continuous [8,44]. Among the human adults, more than 85% of the population was found to be seropositive for AAV. These findings suggested that AAV can establish latent infection. However, it was not known how AAV maintained its latency in the cells until the proviruses in a number of latent AAV cell lines were analyzed by Southern blotting [8,34,44-48]. These early analyses demonstrated that AAV establishes its latent infection by integrating into the host chromosomal D N A via its inverted terminal repeats. The provirus often clusters in the head-to-tail tandem form. No identical provirus integration pattern was revealed by Southern blotting among different cell lines. This led to an early conclusion that AAV integrated randomly in the host genome [1,3,7]. However, in recent studies, some AAV-cellular junction sequences were cloned out of two independent cell lines latently infected with wild-type AAV [49-51]. When these junction D N A fragments were used as probes to characterize the AAV integration site in more latently infected cell lines, it

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was discovered that the proviruses in the majority ( ~ 7 0 % ) of the cells examined were linked to the same cellular flanking sequences [4,50,51], suggesting that AAV integrated into host D N A in a site-specific manner. The cellular sequence, named "AAVSI", was localized on human chromosome 19. Moreover, the AAV proviruses were also directly visualized by in situ hybridization on q l 3.4-19ter of chromosome 19 [51,52]. The mechanism of AAV integration is largely unresolved. In one model, nonhomologous recombination between AAV terminal repeats and host D N A has been proposed based on the fact that patchy homologies exist at AAV and cellular D N A junctions [4,51,53]. The reason why AAV preferentially integrates into the specific region on human chromosome 19 remains a puzzle. In one study [Xiao, X. and Samulski, R.J., unpublished results], cell lines transduced with three mutant AAVneo vectors, namely Rep + Cap , R e p - C a p + , and R e p - C a p - were analyzed by in situ fluorescent hybridization and Southern blotting to see which gene was responsible for the specific integration. The results for all of the mutant AAV-neo vectors showed lower integration specificity into chromosome 19 when compared to wild-type AAV. This study also revealed that other preferential sites also seem to exist on chromosome 2 and on the D-group chromosomes. It should be noted that the viral stocks used to make the AAV-neo cell lines had significant amounts of wild-type AAV contaminants. It is not known if the wild-type AAV had any influence on the specific integration site. Southern blotting and PCR analyses of more AAV-neo cell lines made with wild-type-free R e p - C a p - stock, revealed that less than 5% of the provirus integrated into chromosome 19. This suggests that the recombinant AAV vector significantly lost its specificity for chromosome 19; however, other preferred sites of integration may exist for AAV vectors. IV. Adeno-associated virus as a vector for gene delivery

Because of its unique features, such as lack of pathogenicity and the potential for non-random integration, AAV has recently attracted substantial interest in the field of gene therapy, as a vector to deliver genes to cells in a way that is safe, practical and suitable for commercial development [2,3]. The use of AAV vectors for gene therapy is based on the fact that the AAV genome, when cloned into a plasmid, is still infectious and produces viral particles [5]. The earliest recombinant AAV vectors were constructed by replacing the AAV capsid gene with a Neo r marker gene (Fig. 2). Subsequently, the vector plasmid was cotransfected into the adenovirus-infected cells with a helper plasmid, which could supply the missing capsid function in t r a n s but not itself be packaged into viral particles [54,55]. Initially, two strategies were utilized to make the helper plasmids. One was to insert a large fragment from bacteriophage lambda into the non-essential region of AAV, so that the helper D N A was too big to be packaged, although it was replication-competent [54]. The second way to make the helper plasmid was to delete the majority of the ITR sequences from the AAV genome so that it was deficient in both replication and packaging, but still functional for making Rep and Cap gene products [55]. The recombinant viruses made with such systems demonstrated, in vitro, low transduction efficiencies in the range of 0.5-5% for mammalian cells [2,3]. However, there were two major drawbacks from those vector constructs. First, the

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vectors could only accommodate a foreign gene about 2 kb in size because of the presence of the Rep gene in the vector. In addition, inclusion of the Rep gene was later demonstrated to cause lower transduction efficiency, probably due to the cytotoxicity of its leaky expression. Second, the contaminating wild-type AAV generated through the homologous regions between the vector and complementing helper plasmid was rather high, ranging from 5% to 50% [2,3]. In order to acquire more space for foreign genes, additional recombinant AAV vectors were constructed, which had deletions in both Rep and Cap genes. One of these constructs, named "d13-94", had only about 430 bp of AAV sequences remaining, which consisted of two ITRs (2 x 145 bp) flanked by a 139 bp AAV polyadenylation signal region (Fig. 2). This construct could accommodate a foreign gene of approximately 4.2 kb. However, the possibility of generating wild-type AAV with this construct still exists because of the homology between the 139 bp region of the vector and the helper plasmid [54].

IV. 1. Packaging ,sTstem Ji'ee o/' wild-type A A V In an effort to avoid the formation of wild-type AAV during the growth of the recombinant viruses, a new-generation packaging system was created by completely eliminating the homologous sequences between the vector and the complementing helper plasmid (Fig. 2) [56]. In this system, the vector plasmid psub201 only contains the 1TR plus a 45 bp non-coding sequence, on either end of the AAV genome, after removal of the entire AAV coding region by Xbal digestion (Fig. 2). The foreign gene can be subsequently inserted into this vector plasmid. To construct the helper plasmid, the AAV coding region removed by Xbal digestion from psub201 was placed into a separate plasmid. To each end of the AAV coding region, the adenovirus 5' terminal fragment was added to enhance the AAV gene expression. Since this new helper plasmid, named "pAAV/Ad", does not have AAV sequences homologous to the vector plasmid, the recombinant viruses made with this system

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do not contain wild-type AAV virus contaminants detectable either by Southern blotting or by PCR analysis. Therefore, this packaging system seems to be free of wild-type AAV contamination [56]. Recently, several labs have demonstrated that the 145 bp AAV-ITR is the only cis-sequence required for efficient viral rescue, replication, packaging and integration [Xiao, X. and Samulski, R.J., unpublished results; Muzyczka, N., personal communication]. Additional vectors have been constructed which do not contain any non-ITR sequences from AAV, therefore minimizing the possibility of generating wild-type AAV through homologous recombination and maximizing the capacity of foreign gene sequences that can be accommodated. A lacZ-AAV vector of 5 kb, including the two ITRs, i.e. 106% of wild-type genome size, has been made and can be efficiently packaged into infectious particles, suggesting that AAV has a potential to accommodate a foreign gene of up to 4.7 kb [Xiao, X. and Samulski, R.J., unpublished results]. However, since the current packaging systems still depend on co-transfection of the vector and the helper plasmids, it presents a major abstacle for large-scale preparation. In addition, the recombinant virus yield (usually 105 107 infectious units/ml without concentration) is considerably lower than that of a typical wildtype AAV infection (101°-10 ll infectious particles/ml). The obvious reasons that account for the lower vector titer may be the low efficiency of co-transfection and the inability of the complementing helper plasmid to replicate to a higher copy number. Making a AAV packaging cell line will be very helpful for the application of AAV as a gene delivery vector. Unfortunately, efforts to make packaging cell lines expressing Rep and Cap genes have not been successful mainly due to the cytotoxic effect of Rep gene expression [2,3,57,58]. A HeLa cell line, latently infected with wild-type AAV, was generated as a packaging cell line in which the provirus had impaired ITRs and could not be rescued. As a result, no massive production of wildtype AAV could occur [59]. Nevertheless, the recombinant virus yield was very low from those cell lines (103-104 infectious particles/ml) probably due to insufficient helper functions provided by the low copy number of the provirus. In addition, the wild-type AAV contamination could still occur because of the homology between the vector and the proviruses. Therefore, the ideal AAV packaging cell line should have at least the following features: (a) no homology between the complementing helper genes and the AAV vectors, to minimize wild-type AAV contamination; (b) tightly controlled and highly inducible AAV viral gene expression, especially for the Rep gene, to lower the cytotoxicity and increase the helper function; and (c) limited amplification of the helper genes to augment the helper functions. Currently, numerous strategies have been under investigation, such as placing the Rep gene under highly inducible promoters; selection of conditional Rep mutants such as temperature-sensitive mutants or non-sense mutants; and combination of AAV genes with other viral or cellular amplification systems or incorporating AAV genes with other viral vectors. These studies will eventually lead to a much improved AAV packaging system.

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IV.2. Host range of AA V vectors

AAV displays a very broad range of hosts including chicken, rodent, monkey and human cells [3,34,48,55,60-63]. It is believed that AAV could virtually infect any species of mammalian cells. In the absence of helper viruses, recombinant AAV vectors have been demonstrated to be able to transduce a variety of established tissue culture cell lines with various efficiencies from a few percent to 70% [56,64]. In the cases so far characterized, the vectors existed as integrated proviruses in the host genomes [34,48,56]. AAV vectors has been shown to be able to transduce primary cells very efficiently. In one study, primary bronchial epithelial cells directly isolated from a cystic fibrosis (CF) patient were transduced with an AAV-lacZ vector with an efficiency of 30%, visualized by B-gal staining [Samulski, R.J., personal communications]. AAV vectors have been demonstrated to successfully transduce hematopoietic progenitor cells of rodent or human origin [65] [Samulski, R.J., personal communications]. The transduction efficiencies have reached as high as 90% in individual studies either with AAV-neo or AAV-lacZ vector [Srivastava, A. and Samulski, R.J., personal communications]. In another study, the AAV vector transduction efficiencies were directly compared with a retrovirus vector in human tumor-infiltrating lymphocyte (TIL) cell cultures [Economou, J. et al., personal communication]. The AAV vector has been used to achieve successful transduction after G418 selection in 2 out of 3 experiments. In contrast, the retrovirus vector only successfully transduced TIL cells in 2 out of 25 experiments after G418 selection. Moreover, semi-quantitative PCR of the genomic D N A from the pooled transduced TIL cells has indicated that the copy number for the neo gene introduced by the AAV vector was more than 2 orders of magnitude higher than that of retrovirally-transduced TIL [Economou, J. et al., personal communication]. Although integration of the AAV-transduced genes are expected, no direct characterization has been performed. Another issue involving AAV vectors is whether they can infect non-dividing cells. Although no systematic studies have been done, some evidence suggests that AAV vectors can indeed infect non-dividing cells. For example, an AAV-lacZ vector was injected into various regions of the adult rat brain. At 2 days and at 1 week postvector-injection, numerous blue cells were detected in the brain tissue with B-gal staining whereas no staining was found in the control [Xiao, X. and Samulski, R.J., unpublished results]. In another study, primary human fibroblast cells were infected with wild-type AAV at confluencies of 12%, 25%, 50%, 75% and 100%, respectively. Regardless of cell confluencies, no differences in infection and integration efficiency were observed by Southern analysis, implying that cell division is not absolutely essential for efficient integration [Srivastava, A., personal communication]. IV.3. Expression of transduced genes in AA V vectors

A variety of foreign genes have been inserted into AAV vectors under control of different promoters and have been successfully expressed. The foreign genes include: (a) reporter genes, such as lacZ, CAT, Neo r and luciferase; (b) human cDNAs from gamma- and beta-hemoglobin genes [65,66] and the C F T R gene [25,64,67]; and (c)

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anti-sense R N A [68]. The promoters used to control the gene expression include: (a) viral promoters such as SV40, CMV, RSV-LTR, herpesvirus T K promoter, parvovirus B-19 promoter [3], AAV p5 and p40 promoters [55,64]; (b) human gene promoters such as the gamma-globin promoter [66]; and (c) R N A pol III promoter such as the promoter from the adenovirus VA gene [Samulski, R.J., personal communications]. One example of gene expression using AAV vectors involves the human gammaglobin gene. This gene was inserted into an AAV vector under the control of the globin promoter and a tissue-specific enhancer (LCR site II). When introduced into erythroleukemia K562 cells, the transduced AAV-globin gene exhibited the same basal level of expression as the endogenous globin gene. More importantly, this transduced AAV-globin gene was as highly inducible as the endogenous globin gene by the addition of hemin, a globin gene expression inducer [66]. Another example of tissue-specific expression was shown when the human parvovirus B-19 genome was inserted in the AAV vector. Within that vector, the B-19 genes retained their original tissue specificity and tropism [63]. This suggests that the AAV terminal repeat does not interfere with expression of foreign genes, which is of particular importance for the potential application of the AAV vector as a gene therapy treatment for hemoglobinopathies. Long-term in vivo gene expression has recenty been demonstrated in the lungs of rabbits that received AAV-CFTR vectors via local administration to the airway epithelium. At various time points during a 6-month period, the presence of vector D N A as well as the expression of the C F T R gene from the vector was detected by PCR for D N A and RNA, respectively. In addition, C F T R protein also was detected by Western blot and immunohistochemical staining. No pathological effects were observed in the animals [67]. This is the first example where AAV vectors were demonstrated to be safe and functional in an animal model. V. Advantages and limitations of AAV vectors

There are a number of advantages for using AAV as a vector for gene therapy which are listed below: (1) AAV is a non-pathogenic human virus since no disease has been associated with it. This will minimize the risk to patients if wild-type AAV is generated. (2) The broad host range and lack of tissue tropism, coupled with high transduction efficiency, increases the potential to use AAV vectors to treat a diversity of diseases. (3) The potential non-random integration decreases the probability of insertional mutagenesis. (4) The only AAV sequences in the vector are the ITRs, which are basically transcriptionally neutral and therefore do not interfere with the regulation of foreign genes. (5) There is no immunity to superinfection of AAV so it will be possible to deliver two different genes by co-infection into one cell or to infect the same cell repeatedly. (6) AAV can infect non-dividing cells, although it is not clear if the vector D N A will integrate. The ability to infect quiescent cells makes AAV suitable for many tissue targets such as brain and liver.

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(7) The remarkable stability and relatively high titers of AAV vectors make scale-up and commercialization more feasible. AAV vectors may have some limitations, listed below, that should be considered: (1) The size of the foreign gene that can be inserted into the AAV vector is approximately 4.6-4.7 kb; however, not many candidate genes are eliminated on this basis. (2) The integrated AAV vector potentially could be mobilized if the cells become superinfected with both wild-type AAV and helper viruses. However, if such an event was to occur, it is unlikely that a hyb:id virus, consisting of both the wild-type AAV genome and the foreign gene, would be generated due to the limited packaging capacity of AAV particles. In addition, there is always a significant portion of integrated proviruses that have impaired ITRs resulting from integration and, therefore, could not be rescued. (3) Wild-type AAV infection was reported to interfere with cell cycles and change cell morphology [69,70]; however, the effects are only temporary. No evidence indicates that AAV vectors interfere with cell cycles. In fact it has been reported that when CD4 + and CD8 + lymphocytes are transduced with a AAV-neo vector, the immunological and cytological features of the transduced cell are not altered [71].

VI. Conclusions AAV-2 is a defective, non-pathogenic human D N A virus. The attractive features of high efficiency transduction and stable, non-random integration have made AAV a very promising candidate for developing vectors for gene therapy. The feasibility of using AAV as a safe and effective vector for gene therapy has been demonstrated by numerous studies in which a variety of foreign genes have been successfully delivered and expressed in target cells. Along with the progress of basic biology, improvements of the packaging system and studies in animal models, we anticipate that AAV vectors will be used in clinical studies in the near future.

Acknowledgements The authors wish to thank J.S. Economou, T.R. Flotte, R.J. Samulski, and A. Srivastava for sharing the results of their experiments prior to publication. The assistance of M. Konrad and R. van Kuyk in preparing the manuscript is also greatly appreciated.

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