Birth of a New Therapeutic Platform: 47 Years of Adeno-associated Virus Biology From Virus Discovery to Licensed Gene Therapy
Authors: Terence R Flotte
Journal: Molecular Therapy (2013)

Within American biomedical research, as supported primarily by the National Institutes of Health (NIH) since the post–World War II era, it has been considered axiomatic that investments in discovery research will ultimately lead to the development of novel approaches to diagnostics and therapeutics that will improve the health and well-being of citizens of the United States and the broader world. I reflect here in a historical context on how one such series of investments in discovery has resulted in the emergence of the first licensed human gene therapy product in Europe, namely Glybera.1Burnett JR Hooper AJ Alipogene tiparvovec, an adeno-associated virus encoding the Ser(447)X variant of the human lipoprotein lipase gene for the treatment of patients with lipoprotein lipase deficiency.Curr Opin Mol Ther. 2009; 11: 681-691PubMed Google Scholar,2Bryant LM Christopher DM Giles AR Hinderer C Rodriguez JL Smith JB et al.Lessons learned from the clinical development and market authorization of Glybera.Hum Gene Ther Clin Dev. 2013; 24: 55-64Crossref PubMed Scopus (142) Google Scholar,3Ylä-Herttuala S Endgame: Glybera finally recommended for approval as the first gene therapy drug in the European Union.Mol Ther. 2012; 20: 1831-1832Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar Glybera (alipogene tiparvovec) is a recombinant adeno-associated virus (rAAV) vector with AAV2 inverted terminal repeats (ITRs) encapsidated into AAV1 capsids (so-called rAAV2/1, or, for this purpose, rAAV1), which, when administered intramuscularly, can result in expression of sufficient levels of lipoprotein lipase (LPL) to be considered a safe and effective treatment of LPL deficiency. It was licensed in the European Union in November 2012, 47 years after the near simultaneous discovery of AAV in laboratories at the NIH Bethesda campus and at the University of Pittsburgh.4Blacklow NR Hoggan MD Rowe WP Isolation of adenovirus-associated viruses from man.Proc Natl Acad Sci USA. 1967; 58: 1410-1415Crossref PubMed Scopus (84) Google Scholar,5Blacklow NR Hoggan MD Kapikian AZ Austin JB Rowe WP Epidemiology of adenovirus-associated virus infection in a nursery population.Am J Epidemiol. 1968; 88: 368-378Crossref PubMed Scopus (153) Google Scholar,6Blacklow NR Hoggan MD Rowe WP Serologic evidence for human infection with adenovirus-associated viruses.J Natl Cancer Inst. 1968; 40: 319-327PubMed Google Scholar,7Hoggan MD Blacklow NR Rowe WP Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics.Proc Natl Acad Sci USA. 1966; 55: 1467-1474Crossref PubMed Scopus (320) Google Scholar,8Atchison RW Casto BC Hammon WM Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (560) Google Scholar I trace key milestones in the progress from the discovery of the virus, to its use as a platform for an approved therapeutic product (Figure 1). The emergence of electron microscopy (EM) in the 1960s revolutionized many aspects of biomedical research but perhaps none more so than the discipline of virology. The identification of a simian virus contaminating poliovirus vaccines led to widespread use of EM to examine preparations of partially purified viruses of all types.9Horvath BL Fornosi F Excretion of SV-40 virus after oral administration of contaminated polio vaccine.Acta Microbiol Acad Sci Hung. 1964; 11: 271-275PubMed Google Scholar,10Katz SL Efficacy, potential and hazards of vaccines.N Engl J Med. 1964; 270: 884-888Crossref PubMed Google Scholar This practice led to the discovery of a number of “satellite viruses” that had the ability to replicate efficiently in the presence of other viruses. The application of this process to adenovirus preparations led two groups—one led by M. David Hoggan at the Laboratory of Infectious Diseases of the National Cancer Institute of the NIH Bethesda campus, the other by Robert W. Atchison of the Department of Epidemiology and Microbiology in the Graduate School of Public Health at the University of Pittsburgh—to identify small DNA viruses (20–25 nm in diameter) contaminating cultures of simian and human adenoviruses.7Hoggan MD Blacklow NR Rowe WP Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics.Proc Natl Acad Sci USA. 1966; 55: 1467-1474Crossref PubMed Scopus (320) Google Scholar,8Atchison RW Casto BC Hammon WM Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (560) Google Scholar These particles were proven to be antigenically distinct from adenoviruses and therefore not simply degradation products of the latter. Furthermore, Atchison deduced that “replication of the particles in cell culture was obtained only when they were inoculated simultaneously with adenoviruses. This suggests that these adenovirus-associated particles behave as defective viruses.”8Atchison RW Casto BC Hammon WM Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (560) Google Scholar From that point on, the terms “adenovirus-associated virus” and “adeno-associated virus” were both used for a period of time, with the shorter version ultimately being accepted by the International Committee on Taxonomy of Viruses (ICTV) (http://ictvonline.org). Shortly after their discovery by EM, Neil Blacklow, an infectious disease physician at the NIH working with Hoggan, identified AAV in fresh human tissues from infants and children with adenovirus-induced diarrheal illnesses.4Blacklow NR Hoggan MD Rowe WP Isolation of adenovirus-associated viruses from man.Proc Natl Acad Sci USA. 1967; 58: 1410-1415Crossref PubMed Scopus (84) Google Scholar,5Blacklow NR Hoggan MD Kapikian AZ Austin JB Rowe WP Epidemiology of adenovirus-associated virus infection in a nursery population.Am J Epidemiol. 1968; 88: 368-378Crossref PubMed Scopus (153) Google Scholar,6Blacklow NR Hoggan MD Rowe WP Serologic evidence for human infection with adenovirus-associated viruses.J Natl Cancer Inst. 1968; 40: 319-327PubMed Google Scholar The coinfection by AAV did not substantially alter the clinical course of these infections but showed a trend toward less severe symptoms in coinfected patients. Thus, the concept of AAV being a nonpathogenic virus began to emerge. In fact, a series of subsequent studies showed a tendency of AAV to inhibit the tumorigenicity of other viruses, such as oncogenic adenoviruses, papillomaviruses, and herpesviruses, a property that has yet to be fully explained or explored. Needless to say, the innocuousness of a virus can diminish somewhat the public health demand for its study.11Katz E Carter BJ Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line.Cancer Res. 1986; 46: 3023-3026PubMed Google Scholar,12de la Maza LM Carter BJ Inhibition of adenovirus oncogenicity in hamsters by adeno-associated virus DNA.J Natl Cancer Inst. 1981; 67: 1323-1326PubMed Google Scholar,13Coker AL Russell RB Bond SM Pirisi L Liu Y Mane M et al.Adeno-associated virus is associated with a lower risk of high-grade cervical neoplasia.Exp Mol Pathol. 2001; 70: 83-89Crossref PubMed Scopus (32) Google Scholar,14Hermonat PL Adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer.Cancer Res. 1994; 54: 2278-2281PubMed Google Scholar,15Hermonat PL Plott RT Santin AD Parham GP Flick JT Adeno-associated virus Rep78 inhibits oncogenic transformation of primary human keratinocytes by a human papillomavirus type 16-ras chimeric.Gynecol Oncol. 1997; 66: 487-494Abstract Full Text PDF PubMed Scopus (35) Google Scholar Subsequent study of AAV thus focused not on its clinical importance but rather on the fascinating question of how a virus expressing only two major genes could possibly function. In 1969, a team at the National Institute of Allergy and Infectious Diseases demonstrated that AAV is a single-stranded DNA virus, utilizing an isotopic labeling method (Figure 2).16Rose JA Berns KI Hoggan MD Koczot FJ Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA.Proc Natl Acad Sci USA. 1969; 64: 863-869Crossref PubMed Scopus (106) Google Scholar One of the authors of that article, Kenneth I. Berns, subsequently made a series of pivotal discoveries with regard to the AAV life cycle, including description of its persistent infection in cultured cells, a hairpin self-priming mechanism for AAV DNA replication, and, later, the ability of AAV2 to preferentially integrate within what is now termed the AAVS1 locus on human chromosome 19 (refs. 17Samulski RJ Zhu X Xiao X Brook JD Housman DE Epstein N et al.Targeted integration of adeno-associated virus (AAV) into human chromosome 19.EMBO J. 1991; 10: 3941-3950Crossref PubMed Scopus (667) Google Scholar, 18Cheung AK Hoggan MD Hauswirth WW Berns KI Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells.J Virol. 1980; 33: 739-748Crossref PubMed Google Scholar, 19Berns KI Hauswirth WW Fife KH Lusby E Adeno-associated virus DNA replication.Cold Spring Harb Symp Quant Biol. 1979; 43 (Pt 2): 781-787Crossref PubMed Google Scholar, 20Berns KI Hauswirth WW Adeno-associated viruses.Adv Virus Res. 1979; 25: 407-449Crossref PubMed Scopus (57) Google Scholar, 21Kotin RM Berns KI Organization of adeno-associated virus DNA in latently infected Detroit 6 cells.Virology. 1989; 170: 460-467Crossref PubMed Scopus (111) Google Scholar, 22Kotin RM Siniscalco M Samulski RJ Zhu XD Hunter L Laughlin CA et al.Site-specific integration by adeno-associated virus.Proc Natl Acad Sci USA. 1990; 87: 2211-2215Crossref PubMed Scopus (801) Google Scholar, 23Kotin RM Menninger JC Ward DC Berns KI Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter.Genomics. 1991; 10: 831-834Crossref PubMed Scopus (260) Google Scholar, 24Kotin RM Linden RM Berns KI Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination.EMBO J. 1992; 11: 5071-5078Crossref PubMed Scopus (409) Google Scholar). Interest in AAV biology increased as the novel mechanisms of DNA replication (including replication of the terminal repeats), messenger RNA (mRNA) splicing, and translation of the various versions of the AAV Rep and Cap proteins were elucidated.25Snyder RO Samulski RJ Muzyczka N In vitro resolution of covalently joined AAV chromosome ends.Cell. 1990; 60: 105-113Abstract Full Text PDF PubMed Scopus (123) Google Scholar,26Snyder RO Im DS Muzyczka N Evidence for covalent attachment of the adeno-associated virus (AAV) Rep protein to the ends of the AAV genome.J Virol. 1990; 64: 6204-6213PubMed Google Scholar,27Snyder RO Im DS Ni T Xiao X Samulski RJ Muzyczka N Features of the adeno-associated virus origin involved in substrate recognition by the viral Rep protein.J Virol. 1993; 67: 6096-6104Crossref PubMed Google Scholar,28Im DS Muzyczka N Factors that bind to adeno-associated virus terminal repeats.J Virol. 1989; 63: 3095-3104PubMed Google Scholar,29Im DS Muzyczka N The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity.Cell. 1990; 61: 447-457Abstract Full Text PDF PubMed Scopus (356) Google Scholar,30Im DS Muzyczka N Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization.J Virol. 1992; 66: 1119-1128PubMed Google Scholar,31Li Z Brister JR Im DS Muzyczka N Characterization of the adeno-associated virus Rep protein complex formed on the viral origin of DNA replication.Virology. 2003; 313: 364-376Crossref PubMed Scopus (21) Google Scholar,32Zhou X Zolotukhin I Im DS Muzyczka N Biochemical characterization of adeno-associated virus Rep68 DNA helicase and ATPase activities.J Virol. 1999; 73: 1580-1590Crossref PubMed Google Scholar,32Zhou X Zolotukhin I Im DS Muzyczka N Biochemical characterization of adeno-associated virus Rep68 DNA helicase and ATPase activities.J Virol. 1999; 73: 1580-1590Crossref PubMed Google Scholar,33Kyöstiö SR Owens RA Weitzman MD Antoni BA Chejanovsky N Carter BJ Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.J Virol. 1994; 68: 2947-2957Crossref PubMed Google Scholar,34Trempe JP Carter BJ Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.J Virol. 1988; 62: 3356-3363Crossref PubMed Scopus (88) Google Scholar,35Tratschin JD Tal J Carter BJ Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral Rep gene product.Mol Cell Biol. 1986; 6: 2884-2894Crossref PubMed Scopus (98) Google Scholar,36Owens RA Carter BJ In vitro resolution of adeno-associated virus DNA hairpin termini by wild-type Rep protein is inhibited by a dominant-negative mutant of Rep.J Virol. 1992; 66: 1236-1240PubMed Google Scholar,37Owens RA Weitzman MD Kyöstiö SR Carter BJ Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins.J Virol. 1993; 67: 997-1005PubMed Google Scholar,38Weitzman MD Kyöstiö SR Carter BJ Owens RA Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA.J Virol. 1996; 70: 2440-2448PubMed Google Scholar,39Trempe JP Carter BJ Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation.J Virol. 1988; 62: 68-74Crossref PubMed Google Scholar,40Jay FT de la Maza LM Carter BJ Parvovirus RNA transcripts containing sequences not present in mature mRNA: a method for isolation of putative mRNA precursor sequences.Proc Natl Acad Sci USA. 1979; 76: 625-629Crossref PubMed Scopus (9) Google Scholar,41Beaton A Palumbo P Berns KI Expression from the adeno-associated virus p5 and p19 promoters is negatively regulated in trans by the Rep protein.J Virol. 1989; 63: 4450-4454PubMed Google Scholar,42Berns KI Kotin RM Labow MA Regulation of adeno-associated virus DNA replication.Biochim Biophys Acta. 1988; 951: 425-429Crossref PubMed Scopus (31) Google Scholar During this time, the laboratories of Berns at the University of Florida and Cornell Weill Medical College, Nicholas Muzyczka at the University of Florida and Stony Brook University, and Barrie Carter at the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) published many of the notable findings. The secondary structure of the AAV ITRs made it technically difficult to clone AAV sequences into a bacterial plasmid. This hurdle was overcome in parallel efforts by the teams of Muzyczka (including R. Jude Samulski) at the University of Florida and Barrie Carter at the NIDDK.43Samulski RJ Berns KI Tan M Muzyczka N Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells.Proc Natl Acad Sci USA. 1982; 79: 2077-2081Crossref PubMed Scopus (339) Google Scholar,44Samulski RJ Srivastava A Berns KI Muzyczka N Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV.Cell. 1983; 33: 135-143Abstract Full Text PDF PubMed Scopus (211) Google Scholar,45Laughlin CA Tratschin JD Coon H Carter BJ Cloning of infectious adeno-associated virus genomes in bacterial plasmids.Gene. 1983; 23: 65-73Crossref PubMed Scopus (188) Google Scholar This facilitated the complete DNA sequencing of the AAV2 genome in 1983 by Arun Srivastava,46Srivastava A Lusby EW Berns KI Nucleotide sequence and organization of the adeno-associated virus 2 genome.J Virol. 1983; 45: 555-564Crossref PubMed Google Scholar working in the Berns laboratory at the University of Florida. In addition to making studies of the molecular biology of AAV2 more tractable, the cloning of AAV DNA facilitated the engineering of recombinant AAV2 (rAAV2) vectors, which were likewise nearly simultaneously proven to be viable for gene transfer in mammalian cells in culture by the Muzyczka and Carter laboratories in 1984.14Hermonat PL Adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer.Cancer Res. 1994; 54: 2278-2281PubMed Google Scholar,47Tratschin JD West MH Sandbank T Carter BJ A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase.Mol Cell Biol. 1984; 4: 2072-2081Crossref PubMed Scopus (142) Google Scholar Suddenly AAV was no longer a novelty but rather a potentially useful tool in the emerging field of gene therapy. Early rAAV vectors were hampered by the contamination of preparations with wild-type AAV, a problem that was overcome by Samulski and Thomas Shenk, with their development of non-overlapping plasmid constructs, including a proviral vector plasmid comprising AAV2-ITRs flanking the gene cargo and a helper plasmid expressing Rep and Cap, in trans.48Samulski RJ Chang LS Shenk T A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.J Virol. 1987; 61: 3096-3101Crossref PubMed Google Scholar,49Samulski RJ Chang LS Shenk T Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.J Virol. 1989; 63: 3822-3828Crossref PubMed Google Scholar The development of this relatively wild-type free system enabled rAAV2 production that was sufficiently efficient for testing in vivo gene transfer in animal models. In the late 1980s and early 1990s, efforts by the Cystic Fibrosis Foundation (CFF) pushed forward the first in vivo gene therapy experiments in animals and humans for both recombinant adenovirus (rAd) and rAAV vectors. In the case of rAAV2, the packaging limit of the vector (approximately 5 kb) limited the size of the promoter cassette that could be used for expression of the cystic fibrosis transmembrane regulator (CFTR), which has a coding sequence of 4.4 kb (ref. 50Flotte TR Solow R Owens RA Afione S Zeitlin PL Carter BJ Gene expression from adeno-associated virus vectors in airway epithelial cells.Am J Respir Cell Mol Biol. 1992; 7: 349-356Crossref PubMed Scopus (152) Google Scholar). The use of the AAV2 p5 promoter and the subsequent discovery of cryptic promoter activity from elements within the AAV2-ITR enabled the packaging of CFTR vectors.51Flotte TR Afione SA Solow R Drumm ML Markakis D Guggino WB et al.Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.J Biol Chem. 1993; 268: 3781-3790Abstract Full Text PDF PubMed Google Scholar These vectors were initially used to overcome the CF defect in airway epithelial cells in culture, and then to express CFTR mRNA and protein in vivo in the rabbit airway after endobronchial instillation with a fiber-optic bronchoscope.51Flotte TR Afione SA Solow R Drumm ML Markakis D Guggino WB et al.Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.J Biol Chem. 1993; 268: 3781-3790Abstract Full Text PDF PubMed Google Scholar,52Egan M Flotte T Afione S Solow R Zeitlin PL Carter BJ et al.Defective regulation of outwardly rectifying Cl– channels by protein kinase A corrected by insertion of CFTR.Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar,53Flotte TR Afione SA Conrad C McGrath SA Solow R Oka H et al.Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.Proc Natl Acad Sci USA. 1993; 90: 10613-10617Crossref PubMed Scopus (445) Google Scholar Studies in both cultured airway cells and in the airways of rabbits and rhesus monkeys indicated that rAAV2-CFTR persisted for at least six months in airway cells as an episomal element.54Flotte TR Afione SA Zeitlin PL Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration.Am J Respir Cell Mol Biol. 1994; 11: 517-521Crossref PubMed Scopus (190) Google Scholar,55Afione SA Conrad CK Kearns WG Chunduru S Adams R Reynolds TC et al.In vivo model of adeno-associated virus vector persistence and rescue.J Virol. 1996; 70: 3235-3241PubMed Google Scholar,56Conrad CK Allen SS Afione SA Reynolds TC Beck SE Fee-Maki M et al.Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.Gene Ther. 1996; 3: 658-668PubMed Google Scholar Based on the observed safety in these studies, rAAV2-CFTR was used for the first human gene therapy in November 1995 in the nose and bronchus of an adult with CF.57Flotte T Carter B Conrad C Guggino W Reynolds T Rosenstein B et al.A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease.Hum Gene Ther. 1996; 7: 1145-1159Crossref PubMed Scopus (219) Google Scholar A series of extensions of that original trial, and follow-up trials in the sinuses of CF patients were also completed.58Moss RB Rodman D Spencer LT Aitken ML Zeitlin PL Waltz D et al.Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial.Chest. 2004; 125: 509-521Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar,59Aitken ML Moss RB Waltz DA Dovey ME Tonelli MR McNamara SC et al.A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease.Hum Gene Ther. 2001; 12: 1907-1916Crossref PubMed Scopus (203) Google Scholar,60Wagner JA Moran ML Messner AH Daifuku R Conrad CK Reynolds T et al.A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis.Hum Gene Ther. 1998; 9: 889-909Crossref PubMed Scopus (94) Google Scholar,61Wagner JA Messner AH Moran ML Daifuku R Kouyama K Desch JK et al.Safety and biological efficacy of an adeno-associated virus vector–cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus.Laryngoscope. 1999; 109 (2 Pt 1): 266-274Crossref PubMed Scopus (175) Google Scholar,62Wagner JA Nepomuceno IB Messner AH Moran ML Batson EP Dimiceli S et al.A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies.Hum Gene Ther. 2002; 13: 1349-1359Crossref PubMed Scopus (219) Google Scholar,63Flotte TR Zeitlin PL Reynolds TC Heald AE Pedersen P Beck S et al.Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)–CFTR vector in adult cystic fibrosis patients: a two-part clinical study.Hum Gene Ther. 2003; 14: 1079-1088Crossref PubMed Scopus (196) Google Scholar,64Flotte TR Schwiebert EM Zeitlin PL Carter BJ Guggino WB Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy.Hum Gene Ther. 2005; 16: 921-928Crossref PubMed Scopus (36) Google Scholar Shortly after the report of rAAV2-CFTR gene transfer in the airways, a rAAV2–tyrosine hydroxylase vector was used for in vivo expression in the brains of nonhuman primates as a preclinical approach to therapy for Parkinson’s disease, and rAAV2–epo and rAAV2–factor IX vectors were demonstrated to be effective for sustained expression of erythropoietin in muscle to stimulate red blood cell production in anemia and factor IX in liver for hemophilia.65Snyder RO Miao CH Patijn GA Spratt SK Danos O Nagy D et al.Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.Nat Genet. 1997; 16: 270-276Crossref PubMed Scopus (497) Google Scholar,66During MJ Samulski RJ Elsworth JD Kaplitt MG Leone P Xiao X et al.In vivo expression of therapeutic human genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector.Gene Ther. 1998; 5: 820-827Crossref PubMed Scopus (155) Google Scholar,67Kaplitt MG Leone P Samulski RJ Xiao X Pfaff DW O'Malley KL et al.Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.Nat Genet. 1994; 8: 148-154Crossref PubMed Scopus (914) Google Scholar,68Snyder RO Miao C Meuse L Tubb J Donahue BA Lin HF et al.Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.Nat Med. 1999; 5: 64-70Crossref PubMed Scopus (316) Google Scholar The ability of rAAV2 vectors to express factor IX was subsequently demonstrated in patients as well.69Kay MA Manno CS Ragni MV Larson PJ Couto LB McClelland A et al.Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nat Genet. 2000; 24: 257-261Crossref PubMed Scopus (878) Google Scholar Within the first 10 years of AAV use in humans, trials of rAAV2 and rAAV1 vectors were undertaken for CF, hemophilia B, Batten’s disease, Canavan’s disease, and α1-antitrypsin (AAT) deficiency.57Flotte T Carter B Conrad C Guggino W Reynolds T Rosenstein B et al.A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease.Hum Gene Ther. 1996; 7: 1145-1159Crossref PubMed Scopus (219) Google Scholar,60Wagner JA Moran ML Messner AH Daifuku R Conrad CK Reynolds T et al.A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis.Hum Gene Ther. 1998; 9: 889-909Crossref PubMed Scopus (94) Google Scholar,61Wagner JA Messner AH Moran ML Daifuku R Kouyama K Desch JK et al.Safety and biological efficacy of an adeno-associated virus vector–cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus.Laryngoscope. 1999; 109 (2 Pt 1): 266-274Crossref PubMed Scopus (175) Google Scholar,62Wagner JA Nepomuceno IB Messner AH Moran ML Batson EP Dimiceli S et al.A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with antrostomies.Hum Gene Ther. 2002; 13: 1349-1359Crossref PubMed Scopus (219) Google Scholar,63Flotte TR Zeitlin PL Reynolds TC Heald AE Pedersen P Beck S et al.Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)–CFTR vector in adult cystic fibrosis patients: a two-part clinical study.Hum Gene Ther. 2003; 14: 1079-1088Crossref PubMed Scopus (196) Google Scholar,64Flotte TR Schwiebert EM Zeitlin PL Carter BJ Guggino WB Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy.Hum Gene Ther. 2005; 16: 921-928Crossref PubMed Scopus (36) Google Scholar,69Kay MA Manno CS Ragni MV Larson PJ Couto LB McClelland A et al.Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nat Genet. 2000; 24: 257-261Crossref PubMed Scopus (878) Google Scholar,70McPhee SW Janson CG Li C Samulski RJ Camp AS Francis J et al.Immune responses to AAV in a phase I study for Canavan disease.J Gene Med. 2006; 8: 577-588Crossref PubMed Scopus (194) Google Scholar,71Manno CS Chew AJ Hutchison S Larson PJ Herzog RW Arruda VR et al.AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.Blood. 2003; 101: 2963-2972Crossref PubMed Scopus (599) Google Scholar,72Worgall S Sondhi D Hackett NR Kosofsky B Kekatpure MV Neyzi N et al.Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus expressing CLN2 cDNA.Hum Gene Ther. 2008; 19: 463-474Crossref PubMed Scopus (328) Google Scholar,73Crystal RG Sondhi D Hackett NR Kaminsky SM Worgall S Stieg P et al.Clinical protocol. Administration of a replication-deficient adeno-associated virus gene transfer vector expressing the human CLN2 cDNA to the brain of children with late infantile neuronal ceroid lipofuscinosis.Hum Gene Ther. 2004; 15: 1131-1154Crossref PubMed Google Scholar,74Leone P Shera D McPhee SW Francis JS Kolodny EH Bilaniuk LT et al.Long-term follow-up after gene therapy for Canavan disease.Sci Transl Med. 2012; 4: 165ra163Crossref PubMed Scopus (180) Google Scholar,75Janson C McPhee S Bilaniuk L Haselgrove J Testaiuti M Freese A et al.Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain.Hum Gene Ther. 2002; 13: 1391-1412Crossref PubMed Scopus (204) Google Scholar,76Flotte TR Trapnell BC Humphries M Carey B Calcedo R Rouhani F et al.Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.Hum Gene Ther. 2011; 22: 1239-1247Crossref PubMed Scopus (266) Google Scholar,77Brantly ML Chulay JD Wang L Mueller C Humphries M Spencer LT et al.Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.Proc Natl Acad Sci USA. 2009; 106: 16363-16368Crossref PubMed Scopus (260) Google Scholar,78Brantly ML Spencer LT Humphries M Conlon TJ Spencer CT Poirier A et al.Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.Hum Gene Ther. 2006; 17: 1177-1186Crossref PubMed Scopus (155) Google Scholar,79Flotte TR Brantly ML Spencer LT Byrne BJ Spencer CT Baker DJ et al.Phase I trial of intramuscular injection of a recombinant adeno-associat