TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL. 110, 1999

THE GORDON WILSON LECTURE

FROM BASIC VIROLOGY TO HUMAN GENE THERAPY

KENNETH I. BERNS

GAINESVILLE, FLORIDA

INTRODUCTIONIn the second half of the twentieth century molecular biology and

genetics have constituted the frontier of science, in much the same waythat physics did in the first half ofthe century. During this same periodthere has been great intellectual ferment as the molecular mechanismsunderlying genetics have become increasingly well understood. How-ever, only in the past ten to fifteen years have applications of thisknowledge become available on a large scale. The biotechnology indus-try now has revenues of greater than 10 billion dollars per year and isexpanding exponentially.A great deal of the interest in biotechnology is focused on its appli-

cation to human health care. Current products are primarily humanproteins expressed in and purified from bacteria or yeast, using recom-binant DNA technology. More recently the possibility of developingnaked DNA vaccines has become increasingly likely. The ultimateapplication ofmodern molecular genetics will be the modification ofthepatient's genome to either correct an inherited defect or to render anindividual more able to resist diseases such as AIDS or cancer.The ability to modify the human genome was identified as the top

priority in molecular medicine in a strategic plan for the NationalInstitutes of Health developed when Bernadine Healy was Director.This is still a high priority since the current NIH Director, HaroldVarmus, was a leading member of the advisory committee on molecu-lar medicine when the recommendation was made. It is pertinent tonote, however, that despite input of significant resources to the devel-opment of gene therapy and the initiation of a large number of clinicaltrials, no cures are known to have resulted from these efforts. Indeed,an advisory committee appointed by Dr. Varmus recommended lessemphasis on clinical trials and more on fundamental research de-

Reprint requests can be submitted to: Kenneth I. Berns, MD, PhD, Dean, College ofMedicine, Interim Vice President for Health Affairs, PO Box 100014, Gainesville, FL 32610-0014.

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signed to better understand the molecular mechanisms underlying therequirements which would have to be met to develop successful genetherapy. Quite literally, the major barrier to successful gene therapy isdevelopment of appropriate vectors for delivery of the genes of interest.One promising vector is the human parvovirus adeno-associated virus.Emergence of this virus as a potential vector is a typical story ofserendipity in which extensive fundamental research on the basicbiology and molecular mechanisms of replication of a human virus,originally of little medical interest because of the lack of associationwith any known disease, suddenly becomes of great interest as apotential vector for gene therapy, precisely because of its biology andlack of pathogenicity.

PARVOVIRUSESThe Parvoviridae are small, single-stranded DNA viruses which

infect species ranging from insects to people (1). The virus particles arenon-enveloped icosahedrons with an approximate diameter of 20-26nm. The genome is a linear, single-stranded DNA molecule with achain length of 4-6 kilobases (kb). The Parvovirinae subfamily con-tains three genera of viruses which infect vertebrates (1). Members oftwo of the genera replicate autonomously in dividing cells in cultureand are pathogenic. One of the pathogenic viruses is the human B19parvovirus, the causative agent of erythema infectiosum and aplasticcrises in patients with sickle cell disease. The third genus of theParvovirinae is the Dependoviruses. The members of this genus do notnormally replicate in cells in culture unless the cells are co-infectedwith an unrelated helper virus, either an adenovirus or a herpesvirus;hence, the commonly used name of adeno-associated virus (AAV).These viruses are unusual because half of the viral particles contain aplus polarity, or coding strand, single-stranded DNA molecule and theremaining particles contain the negative polarity strand. A more un-usual characteristic of AAV is the apparently complete lack of patho-genicity. Although various AAV have been isolated from numerousspecies ranging from chickens to man, they have never been implicatedas the natural cause of any disease. Indeed, in several instances theyhave been suggested to protect the host from disease caused by thehelper virus. Sero-epidemiological data have been interpreted to indi-cate that a lack of antibodies against AAV is associated with anincreased incidence of cervical carcinoma (2). This is of interest, be-cause about 90% of the adult population are seropositive for AAV.Thus, AAV represents the rather unusual situation in virology of a

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viral-host relationship which does not appear to harm the host andmay actually be protective. Viruses are by definition parasites; if onedefinition of life is perpetuation of the genome, a way to accomplishthis would be to establish a stable relationship of the viral genome withthe host cell. In this case it would be to the advantage of the virus toprotect rather than harm the cell. AAV has evolved a life cycle that isdesigned to do this (as described below) and it is precisely this featureof the biology of the virus which makes it such an important potentialvector for human gene therapy.

AAV LIFE CYCLE

The AAV life cycle is directed toward the establishment of a stablerelationship between the virus and the host cell (1). The virus accom-plishes this by integrating its DNA into the genome of the host toestablish a latent infection. As long as the host cell is healthy anddividing, the genome is quiescent and there is little, if any, detectableviral gene expression. However, if the cell is stressed by exposure togenotoxic stimuli, which may include ionizing radiation, metabolicinhibitors, or infection by a lytic virus, such as adenovirus or herpes-virus, the AAV genome is activated, rescued and replicated, and prog-eny virus are produced. Our current model for the AAV life cycle, asdescribed above, is consistent with the original observation that AAVwill not undergo a productive infection unless there is a concomitantinfection by a helper virus. We note that all nuclear DNA viruses whichinfect vertebrates will establish latent infections and most people arelikely to be infected by several such latent viruses. The AAV system isunique in that it is rather easy to establish a latent infection in cellculture; this is done by infecting host cells with AAV in the absence ofhelper virus co-infection (3). The existence of latently infected cells inculture has made it possible to perform detailed studies of the molec-ular mechanisms underlying AAV latency. To understand the conclu-sions reached from such experiments it is helpful to review the geneticsand molecular biology of AAV replication (1).The AAV genome contains two open-reading frames (Figure 1) (4,5).

The open-reading frame in the right half of the genome encodes thethree structural proteins, with overlapping amino acid sequences,which comprise the viral capsid. The open-reading frame in the lefthalf of the genome encodes four regulatory proteins, again with over-lapping amino acid sequences. Four proteins are expressed from asingle open-reading frame, because there are two start sites for tran-

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Map units20 40

.... ...... 8.

rr I -lIm--

Rep78_ ~~~~~~~~~~~~~Rep68Rep 52

Rep40

VP 12 3

_____________________________VP2VP3

FIG. 1. The genetic map of AAV is illustrated. Open reading frames, transcripts,introns and expressed proteins are indicated.

scription and both spliced and unspliced versions of the RNAs aretranslated into the proteins (6). These four proteins are known as Repproteins, because frame-shift mutations at most points in the open-reading frame result in the inhibition of DNA replication (4). The twolarger Rep proteins bind to several sites on the AAV genome includingcritical sites in the inverted terminal repeat (ITR, see below) and canfunction as both a helicase and a sequence-specific DNA endonuclease(7). The two proteins have multiple regulatory functions in all phasesof the AAV life cycle. In the absence of a helper virus co-infection, thelarger Rep proteins repress AAV gene expression and inhibit viralDNA replication (8,9). Further they play a significant role in integra-tion of the viral DNA into the host genome to establish a latentinfection. In the presence of a helper virus (permissive conditions forviral replication) the larger Rep proteins are required for AAV geneexpression and DNA replication (8,10). If the helper virus infects a celllatently infected with AAV, the Rep protein plays a significant role inrescue of the viral DNA from the integrated state (11). Thus, the Repprotein controls all steps of the viral life cycle. In addition to its effectson AAV itself, Rep protein is active in regulation of a variety ofheterologous genes. In particular, Rep protein inhibits the expressionof the helper virus. Presumably the helper virus supplies enough

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critical gene expression to render the cellular milieu permissive forAAV replication and transcription; the AAV rep protein then shutsdown the helper to give the replicating AAV a competitive advantage.

If Rep protein represents the trans-active component in AAV regu-lation, the ITR is the cis-active element. The ITR consists of theterminal 145 nucleotides (nts) at each end of the viral DNA (Figure 2)(12). The terminal 125 nts constitute an overall palindrome inter-rupted in the center by two shorter palindromes of 21 nts each (Figure2). When the ITR sequence is folded on itself so that maximum basepairing can occur, a T-shaped structure is formed. The ITR sequence iscritical for correct regulation ofAAV gene expression in the absence orpresence of helper virus; it is required for the successful initiation ofDNA synthesis when helper virus is present and its inhibition other-wise; it is also necessary both for the integration and subsequentrescue of viral DNA from the host genome.

TTTC-GC-GA-TG-CC-GG-CG-Ca-cC-GGCCTCAGT GAGCGAGCGAGCGCGC AGAGAGGGAGTGG CCAA 3'T 11111111 IIlIliliIIlIIIII 11111 lllll l liiG-CCGGAGTCA CTCGCTCGCTCGCGCG TCTCTCCCTCACC GGTTGA GGTAGTGATCCCCAAGGA 5'C-GG-C ~RBS Spacer ThS

c-C;C-G;C-GC-GG-CAAA

FIG. 2. An AAV 1TR is shown. The figure represents the T-shaped structure resultingfrom the palindromic sequence folded on itself to optimize potential base pairing. Thestem contains a rep binding site (RBS) and a terminal resolution site (TRS).

AAV is unique in terms of the integration of its DNA; it is the onlyhuman DNA virus which is known to integrate with high efficiency ata single site in the human genome (13,14). The integration site hasbeen mapped to the q arm of chromosome 19 (19ql3.3-qter) (15). Amajor research objective has been the determination of the specificproperties of the integration site which render it the only location inthe human genome at which preferential integration of the AAV ge-nome occurs. To answer this question a model system for host cellintegration was developed (16). An 8 kb fragment from chromosome 19which contained the integration site was isolated and cloned into a

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shuttle plasmid which could replicate in either bacteria or mammaliancells. This plasmid was used to develop a tissue culture cell line inwhich the specific integration site was separated from its normalcontext within the q arm of chromosome 19. In spite of this separation,AAV was able to integrate into the plasmid containing the integrationsite fragment. Thus, the significant factor in determining site specificitywas the DNA sequence rather than some other structural feature depen-dent on the context ofchromosome 19. We next determined how much ofthe 8 kb fragment was required for site-specific integration. This wasaccomplished by progressively reducing the amount ofthe 8 kb fragmentpresent in the target plasmid until integration ofthe viral DNA no longeroccurred. A minimum required size of 33 nts was found (17).The target sequence was clearly homologous to the AAV ITR (Figure

2). It contained two sets of sequences which seemed likely to be im-portant for AAV-specific integration into chromosome 19 (Figure 3).The first was a binding site for the AAV Rep protein and the secondwas the terminal resolution site (TRS), at which bound Rep proteinwas known to cleave AAV DNA during replication. Genetic studies inthe model system demonstrated that mutations in these two sequencesindeed abolished site-specific integration. Separate biochemical exper-iments supported the genetic studies by showing Rep protein couldbind to the target sequence and cleave the TRS (18). There was aspacer sequence between the two regions defined above; recent exper-iments have indicated that this sequence also may be important inintegration. The target sequence is very similar to the sequence ar-rangement within the AAV ITR, which plays an important role in theinitiation of viral DNA replication. For this reason two groups haveproposed a model which suggests that a polymeric form of Rep proteinlinks the target site to the ITR ofthe viral DNA (17,18). DNA synthesisinitiates on the viral DNA and at some point the elongating DNAstrand switches its template from viral to cellular DNA to effect thefirst step in integration. Determination of the details of integrationawaits the development of an in vitro assay.

TGGG G C T C G G C G C TCOG----GCTGGTRS Spacer RBS

FIG. 3. The AAVSI required target site for AAV site-specific integration on humanchromosome 19. The RBS, TRS and spacer sequences are indicated.

In summary, AAV is unique among human DNA viruses in tworespects. First, it has never been associated with any disease although

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almost 90% of adults are seropositive. Second, AAV establishes latentinfection by integration at a specific site on human chromosome 19. Asdetailed below, these two properties confer significant potential on theuse ofAAV as a vector for human gene therapy.

VECTOR REQUIREMENTSThere are two requirements for human gene therapy: the first is

isolation and characterization of the gene to be transferred and thesecond is an effective way to deliver the gene to the cell. While a largenumber of genes have been cloned and are ready for delivery, we haveyet to develop a successful means of delivery. Two types of deliverysystems may be required; in the first instance transient expression ofa gene may be all that is required or desired. A DNA vaccine or drugactivation gene for cancer chemotherapy would fall in this category. Inthe second instance both long-term and appropriate levels of geneexpression are desirable. It is the second instance which will be thefocus of the comments below. A successful vector must be able toovercome several hurdles: 1) it must be able to enter the cell; 2) thecorrecting gene must be delivered to the nucleus; 3) a stable relation-ship between the gene and the cellular genome must be established; 4)appropriate gene expression must occur; 5) a toxic or inactivatingimmune host response should be avoided; and 6) direct cellular damagemust not occur.The most effective delivery vehicles that we know are viruses, which

have evolved to overcome many of the hurdles listed above. Althoughother delivery vehicles such as liposomes have been developed, to datethe efficiency of DNA delivery possible with viruses has not beenachieved. However, the use of viruses as vectors presents many chal-lenges to be overcome. Most are pathogenic and must be renderedharmless. Most evoke an immune response that complicates their useas a vector. Attenuation of pathogenicity frequently reduces the abilityof the virus to evade the host response and carry out a successfulinfection. Finally, although all DNA viruses establish latent infections,in most instances we do not understand enough about the underlyingmolecular mechanisms to be able to use the latency potential effec-tively for vector purposes.AAV has been a candidate for development as a vector because of its

lack of pathogenicity and because we do have a detailed picture ofhowthe virus establishes latency. However, there are two major limitationsto such use. First, the small size of the viral genome limits the size ofany potential transgene to an upper limit of approximately 4 kb.

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Second, the Rep proteins are potent regulators of gene expression, bothpositive and negative. Although AAV has no obvious pathogenicity, insome experiments in cell culture apparent latent infection has beenreported to have an effect on expression of a variety of cellular genes.For the latter reason, all current AAV vectors being developed for genetransfer lack the rep gene. The lack of rep raises questions about theextent to which the vectors can be expected to integrate into the hostgenome to establish a persistent presence and expression of the trans-gene and also whether such integration, if it does occur, will be at aspecific site in the genome. In cell culture the absence of Rep proteinabolishes site-specific integration (19). However, as outlined below,although there is no evidence of site-specific integration, use ofAAV asa vector has been associated in numerous instances with persistence oftransgene expression.Current AAV vectors are constructed by insertion of the transgene

between two copies of the AAV ITR in a plasmid. The vector constructis either co-transfected with a Rep and coat protein expressing vectorin which the AAV genes are under control of a heterologous promoter(usually an adenovirus promoter) or the transfection is into a cellwhich can be induced to express one or both of the AAV genes requiredfor virion production. Normally adenovirus infection is required toinduce AAV vector production. Because it is necessary to separate AAVvector from the adenovirus, various strategies have been employed toexpress the needed adenovirus helper functions without concomitantadenovirus production. Other technical hurdles to overcome have been1) recombination between the vector construct and the AAV genes toproduce wild-type AAV contamination, 2) low vector yields, and 3)effective purification of large quantities of vector. In spite of theseconcerns, it has been possible to produce sufficient vector to attemptgene transfer in both animal models and in several phase 1 clinicaltrials.

A MODEL SYSTEMExperiments in which the results in animal models are promising

are described by Hauswirth and Muzyczka and associates at the Uni-versity of Florida, UCSF and UCLA (20,21). In experimental modelsystems using mice and rats these investigators have been able toconstruct an AAV vector in which the transgene is specifically ex-pressed in the outer nuclear layer of the retina. The determinant of thespecificity of the site of expression in the retina is the use of the opsinpromoter, which is physiologically specific to the rods and cones, to

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drive the transgene. This was first demonstrated using the greenfluorescent protein as the transgene in a mouse model. There werethree positive results from the experiment. First was the demonstra-tion that the transgene was indeed specifically expressed in the outernuclear layer ofthe retina (which contains the photoreceptor cells) andnot detectably expressed in other retinal layers. Second was the find-ing that a significant fraction (about 5%) of all of the nuclei in the layerwere seen to fluoresce. At the site of injection of the viral vectorvirtually all of the cells were expressing the transgene and the fractionof positive cells was roughly proportional to the distance from theinjection site. Finally, expression was maintained for at least twomonths.These favorable results led to testing this vector in a rat model of

retinitis pigmentosa (RP). RP is a common cause (about 5%) ofhumanblindness and is caused by an autosomal mutation in one copy of thegene for the protein rhodopsin. An AAV vector construct was madeusing the bovine opsin promoter to drive transcription of a ribozymewhich cleaved the mutant mRNA, but not the wild type mRNA fromthe normal allele. In RP rats the cells of the outer nuclear layerprogressively degenerate and vision is affected, similar to human in-herited and sporadic RP. In the model tested, the AAV vector was ableto significantly retard both the decay in function and the physicaldegeneration of the cells of the outer nuclear layer over a period ofnearly a year. Thus, this vector offers considerable promise as a po-tential therapeutic agent against RP.AAV already has been used as a vector in several clinical trials.

Although it has proven to be remarkably non toxic, so far there hasbeen a failure to achieve therapeutic levels of expression of the trans-gene. This may be because of insufficient dose. The results with themodel system described above suggest that it will be possible to useAAV as a vector to achieve human gene therapy.

NEW VECTORSAs noted above, the current generation of AAV vectors does not

contain the rep gene; thus, the problematic aspects of possible Repprotein toxicity are avoided and the space for insertion of a transgeneinto the vector is maximized. However, the ability of the transgene tobe inserted at a specific site on chromosome 19 is sacrificed. The extentto which retention of this capability is desirable is unknown, but we doknow that wild-type AAV does integrate in a site-specific manner andhas not been associated with any known disease. Therefore, it would be

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of interest to develop vectors which had the capability of site-specificintegration. Ideally, the Rep protein would be available to assist insite-specific integration, but would not be present beyond the initialstages of establishing integration of the transgene. This could beachieved either by transporting Rep protein into the cell together withthe transgene or by creating a vector system in which the rep gene wasintroduced separately from the transgene in a vector that could notintegrate into the host cell genome. Although both approaches havebeen tried, neither has been perfected. The latter approach involves inseveral instances use of a larger heterologous viral genome to carryboth the rep gene and the transgene (between two copies of the AAVITR) at separate sites. In this model the larger viral vector will carrythe transgene into the cell; Rep protein will be produced in adequateamounts to induce rescue of the ITR-transgene sequence and subse-quent integration of that sequence at the AAV specific site on chromo-some 19. A potential AAV-baculovirus vector construct has been re-ported (22) and there are active efforts to construct a similar AAV-adenovirus vector.

CONCLUSIONMost adults are seropositive for AAV. Although the virus has not

been implicated as the cause of any human disease, it was of interestbecause of its unusual biology and because of the ease with which virallatency could be established and studied in cell culture. For the tworeasons just stated, the virus has roused considerable interest as apotential vector for human gene therapy. Although it has yet to besuccessfully adapted for this purpose, sufficiently promising resultshave been achieved to justify continued research. And finally the roleof serendipity has been illustrated once again.

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Howley, PM, eds. Fields Virology 3rd Ed., Philadelphia: Lippincott-Raven;1996:2173-2220.

2. Mayor HD, Drake S, Stahmann J, Mumford DM. Antibodies to adeno-associatedsatellite virus and herpes simplex in sera from cancer patients and normal adults.Am J Obstet Gynecol 1976;126:100-104.

3. Hoggan MD, Thomas GF, Thomas FB, Johnson FB. Continuous "carriage" of adeno-virus associated virus genome in cell cultures in the absence of helper adenoviruses.In: Proceedings of the Fourth Lepetit Colloquium. Elsevier/North Holland Publish-ing Co. Amsterdam, 1972;243-249.

4. Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated vi-

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rus type 2 mutants. J Virol 1984;51:329-339.5. Senapathy P, Tratschin JD, Carter BJ. Replication of adeno-associated virus DNA.

Complementation ofnaturally occurring rep mutants by a wild-type genome or an orimutant and correction of terminal palindrome deletions. J Mol Biol 1984;179:1-20.

6. Mendelson E, Trempe JP, Carter BJ. Identification of the trans-acting rep proteinsof adeno-associated virus by antibodies to a synthetic oligopeptide. J Virol1986;60:823-832.

7. Im DS, Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependentsite-specific endonuclease with DNA helicase activity. Cell 1990;61:447-457.

8. Labow MA, Hermonat PL, Berns KI. Positive and negative autoregulation of theadeno-associated virus type 2 genome. J Virol 1986;60:251-258.

9. Labow MA, Berns KI. The adeno-associated virus rep gene inhibits replication of anadeno-associated virus/simian virus 40 hybrid gene genome in cos-7 cells. J Virol1988;62:1705-1712.

10. Ward P, Urcelay E, Kotin R, Safer B, Berns KI. Adeno-associated virus DNA repli-cation in vitro: Activation by a maltose binding protein/Rep 68 fusion protein. J Virol1994;68:6029-6037.

11. Ward P, Berns KI. In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome. J Mol Biol 1991;218:791-804.

12. Lusby E, Fife KH, Berns KI. Nucleotide sequence of the inverted terminal repetitionin adeno-associated virus DNA. J Virol 1980;34:402-409.

13. Kotin RM, Siniscalco M, Samulski RJ, et al. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA 1990;87:2211-2215.

14. Samulski RJ, Zhu X, Xiao X, et al. Targeted integration of adeno-associated virus(AAV) into human chromosome 19. EMBO J 1991;10:3941-3950.

15. Kotin RM, Menninger JC, Ward DC, Berns KI. Mapping and direct visualization ofa region-specific viral DNA integration site on chromosome 19q 13-qter. Genomics1991;10:831-834.

16. Giraud C, Winocour E, Berns KI. Site-specific integration by adeno-associated virusis directed by a cellular DNA sequence. Proc Natl Acad Sci USA 1994;91:10039-10043.

17. Linden RM, Ward P, Giraud C, Winocour E, Berns KI. Site-specific integration byadeno-associated virus. Proc Natl Acad Sci USA 1996;93:11288-11294.

18. Weitzman MD, Kyostio SR, Kotin RM, Owens RA. Adeno-associated virus (AAV) Repproteins mediate complete formation between AAV DNA and its integration site inhuman DNA. Proc Natl Acad Sci USA 1994;91:5808-5812.

19. Kearns WG, Afione SA, Fulmer SB, et al. Recombinant adeno-associated virus(AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalizedepithelial cell line. Gene Ther 1996;3(9):748-755.

20. Flannery JG, Zolotukhin S, Vaquero MI, LaVail MM, Muzyczka N, Hauswirth WW.Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-as-sociated virus. Proc Natl Acad Sci USA 1997;94:6916-6921.

21. Lewin AS, Drenser KA, Hauswirth WW, et al. Ribozyme rescue ofphotoreceptor cellsin a transgenic rat model of autosomal dominant retinitis pigmentosa. Nature Medi-cine 1998;4(8):967-971.

22. Palombo F, Monciotti A, Recchia A, Cortese R, Ciliberto G, LaMonica N. Site-specificintegration in mammalian cells mediated by a new hybrid baculovirus-adeno-asso-ciated virus vector. J Virol 1998;72:5025-5034.