which converts to a double-stranded template after infection, utilizingthe 96% coding capacity of the genome. Gene expression results inproduction of replication (Rep) and structural (Cap) proteins afterinfection. In addition to these AAV gene products, the virus requiresreplication functions from co-infecting helper virus [usually adenovirus(Ad) or herpesvirus] to proceed through AAV replication and a lyticinfection. In the absence of helper virus or helper replication factors,wtAAV DNA integrates as double-stranded DNA into the host cellgenome, where it persists in a latent state. Subsequent infection withhelper virus results in rescue of the AAV from the latent state andproduction of infectious virus. In human cells, the natural host forwtAAV, integration events can occur in up to 70% of the time into aspecific region of chromosome 19 (Ref. 1). While rAAV transgenesalso persist following integration, the site-specificity of integrationappears to depend upon specific DNA endonuclease and helicaseactivities of the wtAAV Rep proteins2, which are generally deleted inrAAV vectors. In designing gene therapy vectors based on AAV, theentire genome of the parent virus can be deleted and replaced withthe transgene of interest, with the exception of the paired 145nucleotide sequences of the inverted terminal repeat (ITR) structures ateither end of the AAV genome (Fig. 1)3. These T-shaped palindromicelements are the only cis-acting sequences required for packaging,integration and rescue of the transgenes that they flank, and also serveas the origins of DNA replication.

Do recombinant AAV vectors have the potential for site-specific integration?The wtAAV is the only eukaryotic virus known to integrate predominantlyin a specific chromosomal location in the host genome, that is, at thehuman chromosome 19q13.3-qter (also called AAVS1)4. This discoveryraised hopes that AAV gene therapy vectors would not only manifestmore persistent transgene expression than did non-integrating vectors(e.g. those based on adenovirus), but might also decrease the theoreticalrisk of insertional mutagenesis (leading to host cell gene disruption ordisregulation) resulting from random integration of delivered genes.Subsequent investigations have shown, however, that the AAV Rep

INTEREST in the ability to exploit recombinant adeno-associatedvirus (rAAV) vectors for gene therapy has encouraged investigationinto the unique biology of this virus. This review considers the perceivedpros and cons surrounding the development of the AAV as a DNA deliveryvector, and discusses recent data shedding new light on these perceptions,suggesting new directions in recombinant AAV gene vector design andapplication (Box 1).

AAV biologyOriginally identified as a ‘defective satellite virus’ found as a contaminantin laboratory stocks of adenovirus (thus, the roundabout designationadeno-associated virus for this small, non-pathogenic Parvovirinae),wild-type AAV (wtAAV) was soon discovered to be defective forreplication in the absence of other ‘helper’ viruses. The AAV life cycleis unique in that AAV infection of a cell might result in either a lyticinfection or persistence of the viral DNA in the infected cell followingintegration into the host chromosomal DNA. The 4.7-kb genome ofthis Dependovirus is packaged as a single-stranded DNA molecule,

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1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1357-4310(00)01810-4

Paul E. Monahan* MDAssistant Professor, Pediatrics

Division of Hematology/Oncology and Gene Therapy Center,University of North Carolina at Chapel Hill School of Medicine,

CB#7352, Thurston-Bowles Bldg, Chapel Hill, NC 27599, USA.R. Jude Samulski PhD

Professor, Department of Pharmacology

Director, Gene Therapy Center

University of North Carolina at Chapel Hill School of Medicine,Chapel Hill, NC 27599, USA.

Tel: 111 919 966 1178Fax: 111 919 966 0907

*e-mail: Paul_Monahan@med.unc.edu

Adeno-associated virus vectorsfor gene therapy: more pros than cons?

Paul E. Monahan and R. Jude Samulski

Gene therapy vectors based on the adeno-associated virus (AAV) are being developed for a wideningvariety of therapeutic applications. Enthusiasm for AAV is due, not only to the relative safety of thesevectors, but also to advances in understanding of the unique biology of this virus. This review examines anumber of long-standing concerns regarding the utility of AAV for gene transfer in light of many newinsights into the biology, immunology and production of AAV.

proteins are vital to bridge the AAV ITR and the ch-19 target locus andmediate wtAAV site-specific integration. At present, the specificmechanism(s) remains unknown but appears to involve Rep-mediatednicking of chromosome 19 (Chr 19) as well as Rep interaction with aRep-responsive Chr 19 origin of replication5,6. rAAV vectors, whichlack the Rep proteins, appear to employ host cellular factors to integratevia the ITR sequences, but do so into heterogeneous spots throughoutthe host genome7. rAAV vectors, therefore, have all the cis elementsrequired for integration, which can be carried out by host factors, buttargeting is lost in rep-minus constructs as compared to wtAAV.Transgene persistence in vivo in DNA concatamers (double-strandedtandem repeats of input sequence) was described originally inhigh molecular weight (HMW) DNA from rAAV-transduced muscleand assumed to result from integrated copies8,9. Nevertheless, therelative importance of integrated versus episomal vector sequences forHMW-derived persistent expression from transduced muscle hasremained unclear. More definitive evidence for integrated rAAVsequences in transduced tissues has been suggested recently byfluorescent in situ hybridization of vector DNA on metaphase chromo-somes of transduced hepatocytes10; subsequent isolation and

characterization of rAAV vector–cellular DNA integration junctionsequences from mouse liver provide confirmation of persistence viaintegration in this tissue11.

Nevertheless, rep(2) rAAV vectors lack the Chr-19-tetheringfunction of Rep protein to facilitate site-specific integration. Theincorporation of a truncated and RU486-regulatable Rep protein into arAAV vector has been described recently, with restoration of integrationat the Chr-19 site in tissue culture12. The ability to regulate the degreeof Rep expression will probably be an essential safety feature of thisstrategy, as abundant rep gene expression is toxic in many cells, owing,at least in part, to the ability of Rep to downregulate expression froma variety of promoters4. In addition, engineering wtAAV-like site-specific

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Figure 1. Wild-type adeno-associated virus (wtAAV) life cycle and genomicstructure. (a) Biphasic life cycle of the wtAAV. (ai) Latent infection. In theabsence of helper virus to supply required replication functions, the wtAAVestablishes a state of latency following infection of the cell. The virus genesequences insert, via the inverted terminal repeats (ITRs) that flank the virusgenome, into the host cell chromosomal DNA and persist in a quiescentstate. (aii) Lytic infection. AAV can proceed through a lytic infection if coincidentinfection with helper virus (adenovirus or herpesvirus) occurs. Alternatively,if helper virus superinfects a cell carrying latent AAV, the AAV genomerescues from the latent state and proceeds to replicate and complete a lyticinfection. wtAd and wtAAV are produced. (b) Schematic of the wtAAVgenome and the elements of a recombinant AAV (rAAV) vector. The wtAAVcoding sequences for the non-structural (Rep) and structural (Cap) proteinsare each encoded by a single open reading frame. These genes can beentirely deleted from rAAV vectors, leaving 96% of the packaging capacity ofthe virus for the therapeutic gene sequences and regulatory elements of thegene expression cassette. The ITRs are retained in the rAAV, and are vital tovirus packaging, second strand synthesis, and integration. Arrows indicatepromoters; pA, polyadenylation signal.

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Molecular Medicine Today

Box 1. AAV vectors: pros, cons and recent insights

Pros:• Integrating vector, with potential for persistent expression

following integration.• Efficiently transduces a wide range of host cells.• Non-pathogenic virus, minimal cell-mediated immune

responses.Recent insights:

• Persistence via episomal and integrated transgenes.• Site-specificity absent for most vectors; might be restorable,

lowering risk of insertional mutagenesis.• Narrowing the target-cell range could be advantageous for

tissue-specific applications.• Mechanisms of immune evasion proposed and impact of

anti-serotype antibodies being explored.• Heterodimer vectors might effectively double capacity.

Cons:• Small packaging capacity (limits size of transgenes that can

be delivered).• Integrating vector carries risk of insertional mutagenesis.• Prevalence of seropositivity of antibodies against wild-type

AAV: humoral immune responses against vector.• Difficult to produce sufficiently high titer (as required for

human clinical trials).Recent insights:

• Site-specific integration, if it occurs, might interrupt a muscle-specific DNA region.

• Impact of anti-serotype antibodies: might limit efficacy of initial treatment or ability to re-administer gene therapy vector.

• Potential risk of antibody responses versus transgene.• Purification based on identified AAV receptors: rAAV packaging

cell lines.

integration into rAAV vectors might involve specific risks. The AAVS1integration site on Chr-19 has been linked recently to the TNNT1 gene,encoding troponin I isoforms normally expressed in slow skeletal muscle13.Investigations in latently infected cell lines have demonstrated integratedwtAAV sequences disrupting the TNNT1 gene. Whether this eventwould represent a risk in (multinucleated) muscle cells or elsewhere issubject to investigation. While the reader is referred to discussions ofinsertional mutagenesis and germline transmission that are beyond thescope of this review14–16, the burden of investigators will be to provethese very small risks approach zero, especially when proposing rAAVapplications for generally non-lethal conditions (e.g. hemophilia).

Will a wide range of target cells prove targetable with rAAVvectors?rAAV vectors have proven able to transduce a wide range of tissuetypes, leading to gene expression in lung17, neurons18, eye19, liver20,muscle21, hematopoietic progenitors22, joint synovium23, endothelialcells24 and gut25. In fact, initial in vitro rAAV transduction experimentsin primary or immortalized cell lines suggesting that dividing cells aretransduced more efficiently than are non-dividing cells provedmisleading. Most recently, detailed examination of in vivo transducedmouse liver demonstrates equivalent rAAV transduction in dividingand non-dividing hepatocytes; expression is not augmented byprogression through the cell cycle or cell division26. rAAV vectorshave defined a particular niche because of their in vivo efficiency inrelatively terminally differentiated tissues, such as skeletal musclemyotubes and mature neurons. It is speculated that the single-strandedAAV is able to take advantage of unscheduled DNA repair activity inrelatively transcriptionally quiescent cells to facilitate AAV replication–amplification (resulting in second-strand synthesis), as appears tohappen in rAAV-infected tissue culture cells challenged with genotoxicagents, such as ultraviolet light, hydroxyurea and heat shock.Nevertheless, not all non-dividing cells are efficiently transduced(e.g. respiratory epithelium).

Strategies for improving rAAV gene transfer must address at leastthree requirements for in vivo transduction of a given target cell. Theseinclude: (1) AAV interaction with target cell receptors (and putativeco-receptors), leading to attachment, internalization27 and nucleartransport; (2) efficient conversion to a double-stranded template; and(3) adequate expression from the AAV–transgene construct, whetherepisomal or integrated. Some recent insights into AAV biology provideexamples of the potential to improve rAAV transgene delivery.

AAV interaction with target cell receptorsThe recent description of heparan sulfate as a receptor for rAAV (Ref. 28)promptly led to an examination of the heparan sulfate expression onskeletal muscle cell membranes. Unexpectedly, transduction withrAAV vectors was found to be markedly more efficient in mouseslow-twitch muscle fibers than in fast-twitch fibers21. Further examinationrevealed that slow myosin heavy chain isoform and heparan sulfateproteoglycan colocalized in skeletal muscle, demonstrating preferentialrAAV transduction of predominantly slow-twitch fibers (Fig. 2).

Additional efficiency of tissue targeting is being achieved as AAVcapsid biology and the full range of AAV serotypes are characterized.Although the AAV-2 serotype has been used for most vectors to thispoint, there are six primary isolates of AAV (AAV 1–6). Sequencedivergence in the capsid genes implies that the tissue tropisms ofdifferent serotypes will differ. For example, while the sequencehomology in the capsid region between AAV2, AAV3 and AAV6 is

.80%, comparison of each of these to AAV5 capsid gene yields only51–59% identity29. AAV2 infection and transduction of cells (presumablydependent upon interaction with the cellular receptor heparan sulfate)is severely inhibited by preincubation of the virus with heparin, butAAV5 transduction is only mildly competed by heparin, implying adistinct mechanism of uptake30. Comparisons of differing in vivotransduction by varying serotypes have been reported recently in thebrain18, the airway31 and in hematopoietic cells32. For example, strikingdifferences following delivery to brain parenchyma were seen, withAAV4 transducing exclusively ependymal cells, AAV2 transducingonly neurons, and AAV5 transducing 130–3000 times more cells thandoes AAV2, including neurons and astrocytes18.

Efficient conversion to a double-stranded templateLong-term (three-week) cultures of primary human skeletal musclecells show that expression of transgene after rAAV infection risessteadily, in parallel with the conversion of input monomer virus todouble-stranded form. This early rise in expression is independent ofconversion to a HMW form33. Data for a molecular mechanism tosupport these observations have now been reported. A cellular Tyrphosphoprotein has been described recently [the single-stranded D

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GlossaryAdeno-associated virus (AAV) – Any of a number of small, single-stranded DNA animal viruses of the genus Dependovirus of thefamily Parvoviridae; the term is often used to refer to the membersof the genus for which humans might be a host (AAV1–6).

Episome – Non-integrated circular, double-stranded DNA moleculeswith the capacity to integrate into the chromosome of the host cell.

Insertional mutagenesis – The disruption or abnormal regulationof genes within a cell as the result of integration of exogenous DNA(e.g. gene therapy vector sequences); potential risks includeoncogenesis and loss of cell function.

Integration – Insertion into a host genome of viral or other DNAsequences as a region covalently linked on either side to the hostsequences.

Neutralizing antibody – An antibody that, in binding to a viralgene therapy vector, reduces or eliminates its ability to infect andtransduce cells.

Parvovirinae – Viral subfamily of small, single-stranded DNA animalviruses, composed of viruses (in the genera parvovirus anderythrovirus) which have the ability to independently replicate afterinfection of a cell, and viruses of the genus dependovirus, whichrequire co-infection by unrelated helper virus for replication.

Serotype – Subtype of a species that is based on antigenicdifferences.

Transduction – In the context of gene therapy, the transfer ofeukaryotic cellular genes by a gene therapy vector, resulting inexpression of the delivered gene product.

Transgene – New DNA sequence introduced into host cell by genedelivery vector; in the context of gene therapy, the transgene oftenencodes a therapeutic protein or a marker that can be monitored.

sequence-binding protein (ssD-BP)]34 which binds the AAV ITR andmight regulate synthesis of the complementary strand, suggestingthat strategies to augment or accelerate the presentation of a double-stranded template for transcription will increase transduction by AAVvectors. Analysis of the phosphorylation state of the ssD-BP in a panel oftissues demonstrated the highest dephosphorylated : phosphorylated ssD-BPratio in skeletal muscle, brain, lung and liver, consistent with the efficiencyof transduction by AAV seen in these tissues. The phosphorylated formpredominates in the spleen and thymus, organs the same investigatorsdemonstrated to be transduced less efficiently by AAV vectors34.

Expression from the integrated or episomal AAV/transgene sequencesStudies establishing the potential for persistent in vivo expression fromrAAV have characterized the fate of the input DNA, demonstrating that,in the first weeks post-infection, the great majority of the input single-stranded DNA sequences (rAAV genome copies) disappearfrom infected muscle8 and liver10. Nevertheless, it is precisely duringand immediately after this time that transgene expression levels increase most rapidly, correlating with the appearance of transgenecopies in double-stranded DNA (Refs 9,10,33). Persistence of expres-sion, however, correlates with conversion to HMW form, which couldoccur by amplification and concatamerization of input sequences.Alternatively, there is recent in vivo evidence in muscle and in liverthat intermolecular joining of independent viral genomes occurs,yielding integrated concatamers or circular episomes of inputsequences26, 35. Circular episomes could serve as intermediate structuresthat later integrate, or could themselves be templates for episomalexpression independent of integration (Fig. 3a). At present, the

molecular fate of AAV vectors required for efficient gene expression isstill under investigation.

Does rAAV actually evade host immune responses?Enthusiasm for rAAV vectors has always been driven by the assumptionthat vectors based on a completely non-pathogenic virus (wtAAV) shouldprovide the greatest degree of safety. Public demand for safety has neverbeen higher, as more gene therapy clinical trials appear. Nevertheless,care must be taken when implying rAAV is equivalent to wtAAV. Theburden of infectious particles being delivered (often .1013 vectorgenomes) to localized sites (e.g. into a single joint space or into brainparenchyma) create immune challenges that are hard to compare withnatural AAV infection of the upper respiratory tract in humans.Non-human primate studies involving endotracheal delivery of rAAV tospecific segments of the macaque lung have shown a remarkable lack ofinflammation, as assessed by tissue pathology, pulmonary functiontesting and bronchoalveolar lavage (BAL) with cytokine assays and cellcounts of BAL fluid36. rAAV administered to mouse muscle (in directcomparison to an Ad vector) does not elicit a cell-mediated immuneresponse, but is followed by the development of high-titer antibodiesagainst the AAV capsid, which are neutralizing for infection8,37. Theobservations that antigen-presenting cells (APCs) in some tissues (e.g.muscle) are not efficiently transduced by rAAV, do not express transgeneproduct, and do not proliferate38–41, suggest a possible explanation for lackof cellular immune responses. These observations have not been uni-versal, however; both the CTL response and the humoral response tothe delivered gene depend on a number of variables, including the natureof the transgene, the route and site of injection, the age, health andimmunological background of the subject, the degree of contaminationwith helper virus or helper virus proteins42, and even the maturationstate of APCs exposed to rAAV administration41. One immune responsethat is reliable is the development of antibodies against the capsid of thevector. While the development of gene therapy vectors has been basedprimarily on the AAV2 serotype, six serotypes have been isolated fromprimate samples. In the general population, antibodies to AAV arefound in up to 80% of adults, with antibodies against the AAV2serotype being the most common. These individuals are commonlyreferred to as seropositive for having been exposed to AAV infection.A minority of these individuals and their antibodies (detected byELISA) are able to neutralize in vitro AAV infections22,43. The effect ofthese antibodies on gene delivery with rAAV has yet to be determined; itis notable that two patients treated with rAAV for hemophilia B appearto demonstrate transgene expression despite pre-treatment neutralizingantibody (NAb) titers of 1:100 and 1:1000 (Ref. 44). A new area ofintense interest will be the determination of whether re-administration ofrAAV vectors will be required, and if so, whether the use of alternativeserotypes or selective capsid modifications will circumvent antibodyresponses to specific AAV capsid elements. One might suspect thatshared antigens of the AAV capsids could lead to globally cross-reactivegroup antibodies. For instance, the development of anti-AAV2 NAbsinterferes with liver-directed administration of rAAV1 as well as rAAV2vectors45. Nonetheless, the ability to augment expression using alternativeserotype rAAV following initial AAV2 treatment has been shown in vivo inthe mouse lung (AAV6 following AAV2)17 and muscle (AAV1 after AAV2)45.

Is packaging size of wtAAV a true constraint to delivery oflarger genes?Despite the attractive aspects of persistence and relative safety thatrAAV vectors offer, many clinical applications have appeared strictly

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Figure 2. AAV transduction efficiency is dependent on tissue expression ofreceptors. (a) Skeletal muscle infected with rAAV/beta-galactosidase demonstratesexpression (cells staining blue with LacZ) in discrete cells in muscle bed, withno staining in adjacent myocytes. (b) Cells of slow muscle phenotype asdemonstrated by slow myosin isoform staining correspond precisely withtransduction by rAAV/beta-gal. (c) Immunofluorescent staining for slowmyosin isoform corresponds precisely with myocytes (d) that demonstratecytoplasmic (as well as membrane) overexpression of heparan sulfateproteoglycan (HSP). Photographs courtesy of Johnny Huard, University ofPittsburgh, PA, USA.

off-limits because of what seemed like the inviolable packaging limit(,5 kb) imposed by the size of the wtAAV (Ref. 46). While 80% of allcDNAs fall within the 3–6-kb size range, rAAV delivery of genesnearing or exceeding 5 kb in size [including some that are the objectsof intense investigation, e.g. dystrophin, factor VIII and the cysticfibrosis transmembrane conductance regulator (CFTR)] has beendisappointing. Strategies are being explored to expand the rAAVpackaging capacity, including, the development of hybrid viral capsidstructures (e.g. parvovirus B19/AAV hybrids47), the use of very smallpromoters or promoter activity derived from the AAV ITR48,49, and

heterodimerization of separate rAAV vectors. Heterodimerization ofseparate rAAV vectors takes advantage of a unique property of wtAAVbiology, namely, expression from latent AAV proviral DNA existing inhead-to-tail concatamers4 (Fig. 3a). Recently, three separate groupshave taken advantage of the inherent ability of AAV to form head to tailDNA concatamers, with a strategy that bridges the 59 and 39 segmentsof a split expression construct through paired rAAV inverted terminalrepeats50–52 (Fig. 3b). The ITRs are removed with intronic sequencesduring mRNA processing of the concatamer, resulting in productionof a single large mRNA. Separate rAAV vectors, encoding a promoter/

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Figure 3. Possible mechanisms of persistence of AAV genome following infection. (a) After release from the virus capsid, the single-stranded genome undergoessecond DNA strand synthesis and can persist integrated into the host cell chromosome. This mode of persistence generally involves amplification of the AAV genome(ai); restriction enzyme digestion of integrated DNA (black arrows) yields unit-length copies of AAV DNA, suggesting tandem repeats of virus DNA existing as head-to-tail concatamers. Alternatively, input AAV sequences can persist, either before or in lieu of integration, in episomal concatamers (aii), formed either by amplifi-cation of AAV genomes or by intermolecular joining of separate input virions in head-to-tail orientation. (b) Heterodimer rAAV vector strategies developed based onmechanisms of persistence effectively double the size of DNA sequences that can be delivered. AAV genome persistence as head-to-tail concatamers (integrated orepisomal) is exploited to deliver two halves of a large gene on separate vectors. The inclusion of splice donor (SD) and splice acceptor (SA) sites on the respectivevectors allows the intervening ITR sequence (following recombination at the shared ITR sequences) to be spliced, creating a complete gene. Alternatively, the transgenecan be included on one vector, with the second vector devoted to transcriptional regulatory elements (TRE) such as enhancer and promoter elements (ENH/Pro), whichwill act to increase transgene expression once intervening ITR sequences are spliced from the messenger RNA (mRNA) of the concatamer transcript.

59half of erythropoietin (epo) gene and39end/polyA, respectively, when deliveredtogether to the muscle of rats, protectedthe animals from Epo-deficient anemia in theface of experimentally induced chronic renalfailure51. The net effect of the heterodimer(or ‘trans-splicing’) strategy is to double (toalmost 10 kb) the size of transgene andtranscriptional elements that rAAV vectorscan deliver in vivo.

Will it be possible to produce rAAV tohigh enough titer for humanapplications?New insights into wtAAV biology are alsohelping to address limitations in the productionof high-titer purified rAAV. These limitationshave included: (1) the requirement for helpervirus for AAV production, with the resultantrisk of helper virus contamination; (2) thedependence on purification techniques, suchas density gradient centrifugation over cesiumchloride, which saturate the virus with potentiallytoxic materials; and (3) inefficient productionfrom early generation producer cell lines.Classically, rAAV production involves supplying human cell lines with the AAV rep and cap gene functions in trans from a plasmid, along with a non-homologous AAVITR–transgene plasmid which comprises thevector, and live adenovirus for helper functions(Fig. 4a). Production using transfection-onlytechniques with no live input virus has beendeveloped. Supplying the adenovirus E2a,E4orf6, and VA helper functions from a plasmidallows propagation of rAAV in HEK 293 cellsin triple- or double-transfection productionschemes53,54 (Fig. 4a). rAAV production via eitherof these strategies benefits additionally fromthe recent identification of cellular ligands forAAV-2, including heparan sulfate proteoglycanand co-receptors aVb5 integrin and humanFGF receptor (Refs 28,54–56), which hasled to the use of affinity matrix chromatographicpurification of rAAV free of cesium chlorideor helper virus contamination57. Thesepurification gains should reduce contaminationsufficiently to permit a move away fromtransfection-dependent techniques in thenear future, and towards recently describedproducer cell line strategies that yield rAAVfollowing simple infection with complement-ing hybrid helper adenovirus58 or herpesvirus(HSV)59 (Fig. 4b).

Prospects for the futureIn actuality, the question is no longer ‘Will itbe possible to produce rAAV to high enoughtiter for human applications?’ Human clinical

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Figure 4. Strategies for producing recombinant adeno-associated virus (rAAV) vectors. All strategies dependon AAV inverted terminal repeat (ITRs) flanking the therapeutic gene of interest (so as to act in cis). Theadditional rep and cap genes of AAV can be supplied separately (in trans), along with a source of helper virusgene sequences, to direct replication and packaging of the vector sequences. (a) Strategies requiringtransfection: classical (ai) tissue culture cells are infected with helper virus [adenovirus (Ad) or herpesvirus]to supply necessary replication functions for which the dependent virus AAV is defective; the cells aresubsequently transfected with circular plasmid DNA supplying the AAV rep/cap on one plasmid and theITR/transgene sequences on a separate and non-homologous plasmid. Upon harvest of the rAAV, contaminatinghelper virus (Ad) must subsequently be removed by various purification techniques, such as densitygradient centrifugation over cesium chloride. Triple-transfection (aii) possibility of wild-type contaminationof vector stocks is eliminated by incorporating only the helper replication sequences of the helper virus ona plasmid, which is simultaneously transfected with the vector AAV plasmid and a plasmid encoding the AAVRep and Cap proteins. (b) Producer cell line-dependent strategies. (bi) A producer cell which stablyexpresses Rep and Cap is infected with wtAd virus and a hybrid adenovirus (Ad). The hybrid Ad containsthe therapeutic gene of interest flanked by the AAV ITRs, all flanked by the Ad TR sequences. Ad mustsubsequently be removed from rAAV stock by purification. (bii) A producer cell line stably transformed withthe AAV ITR/transgene expression cassette is infected with a recombinant HSV (rHSV-1) which supplies HSVhelper functions and AAV Rep/Cap in trans, but is crippled for HSV replication. The rHSV-1 is not replication-competent, and specific purification steps to remove contaminating rHSV-1 are not required.

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trials for cystic fibrosis and for hemophilia B have been enrollingpatients since 1996 and 1999, respectively. While doses on the cysticfibrosis trials have been modest, the hemophilia trial delivers quitelarge doses (up to 1013 vector genomes kg21). Importantly, over 50humans with cystic fibrosis have now received rAAV-CFTR vectors tothe airway, and seven humans have received rAAV/Factor IX vectors tothe muscle, with no reported local or systemic toxicity or (in thehemophilia trial) vector spread to germ cells. While comprehensivereviews exist of the current scope of clinical conditions for whichrAAV vectors are being developed43,60, it is likely that clinical trialsinvolving delivery to the central nervous system (Canavan disease),liver (hemophilia B), as well as additional trials in muscle and airway(a-1-antitrypsin deficiency) will commence in the near future.Investigators must continue to explore critical preclinical evaluation ofrisks in the age of clinical trials. Without a doubt, new questions willappear regarding how best to exploit and respect the unique biologicproperties of AAV that recommend, now more than ever, the developmentof rAAV gene therapy vectors.

Acknowledgements. We gratefully acknowledge Jack Cornell and Joseph Rabinowitz for

illustrative assistance and Stuart Gold for valuable comments. Paul Monahan receives

research support from the National Hemophilia Foundation.

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The outstanding questions

• Will observations made in animal models regarding infectivity,transduction, expression, and persistence of recombinantadeno-associated virus (rAAV) be predictive of responses inhumans (the natural host of the virus)?

• Will repeated administrations of rAAV vectors be necessaryor feasible?

• Does prevalence of seropositivity for antibodies againstwild-type (wt)AAV in the general population limit the utilityof rAAV for gene transfer?

• What are the mechanisms for virus entry at the cytoplasmicand nuclear membranes and how will understanding of thesesteps lead to improvements in cell targeting and transductionefficiency?

• What will be the ultimate role for rAAV vectors of alternativeserotypes (AAV1–6)?

• Will production of rAAV be able to move completely awayfrom cell-based and helper virus-based systems?

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38 Jooss, K. et al. (1998) Transduction of dendritic cells by DNA viral vectors directs

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39 Hernandez, Y. et al. (1999) Latent adeno-associated virus infection elicits humoral

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45 Xiao, W. et al. (1999) Gene therapy vectors based on adeno-associated virus type 1.

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46 Dong, J-Y. et al. (1996) Quantitative analysis of the packaging capacity of recombinant

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47 Ponnazhagan, S. et al. (1998) Recombinant human parvovirus B19 vectors:

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What you missed in the October issue of MMT

Protein aggregation in Huntington’s and Parkinson’s disease: implications for therapyE.E. Wanker

Secretases as targets for the treatment of Alzheimer’s diseaseM. Citron

RAS inhibitors: potential for cancer therapeuticsY. Kloog and A.D. Cox

X-linked severe combined immunodeficiency: from molecular cause to gene therapy within seven years

W.J. Leonard

Murine models of malignant melanomasM.K. Tietze and L. Chin

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