AAV's Golden Jubilee
Authors: Nicholas Muzyczka, Kenneth I Berns
Journal: Molecular Therapy (2015)

The paper recently published in Nature by Gardner et al.1Gardner MR Kattenhorn LM Kondur HR von Schaewen M Dorfman T Chiang JJ et al.AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.Nature. 2015; 519: 87-91Crossref PubMed Scopus (215) Google Scholar marks yet another milestone in the development of adeno-associated virus (AAV) as an effective gene delivery vehicle, and is an appropriate way to highlight the fiftieth anniversary of the discovery of AAV. Using simple intramuscular injections in primates, Gardner et al. demonstrated that they can use AAV to express a synthetic hybrid antibody-receptor decoy that has neutralizing capacity for a broad range of currently circulating HIV strains. The injected primates were protected against HIV challenge for up to 10 months. They suggest that this approach may be a novel and effective way of providing immunity to HIV. AAV was discovered 50 years ago as a contaminant of adenovirus preparations grown on monkey kidney cell cultures.2Atchison RW Casto BC Hammon W Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (556) Google Scholar The viral particles were ~26 nm in diameter (Figure 1), and it was apparent from the start that it was a distinct virus that could be propagated only in the presence of a helper virus, either adenovirus or herpesvirus. Infection of cells with AAV alone produced no cytopathic effect, and no human disease has ever been associated with AAV infections. Early work in cell culture also suggested that AAV could cause a latent infection that could be rescued by infection with the helper virus. It was commonly assumed at the time that latency was achieved by viral integration. Because it caused no disease, AAV received relatively little attention by the medical community and only a handful of laboratories pursued the unusual biology of this single-stranded DNA virus. However, when the discovery of restriction enzymes made cloning possible, the finding that AAV could persist in a latent state and caused no disease made it a viable candidate for development as a gene therapy vector. AAV's requirement for a helper virus for propagation provided an additional layer of safety against potential spread of recombinants in the human population, a major concern at the time. Because AAV did not form viral plaques on its own, the first problem was to develop a genetic system. This was solved by Muzyczka's group,3Samulski 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 (337) Google Scholar who cloned the intact wild-type viral genome into a bacterial plasmid and showed that the plasmid itself was infectious and would produce virus. The infectious plasmid, coupled with the complete DNA sequence of AAV,4Srivastava 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 allowed investigators to do a detailed genetic mapping of the AAV coding regions and the inverted terminal repeats (ITRs). Two major open reading frames were identified, rep and cap—rep encoding proteins needed for DNA replication and packaging, cap encoding the three capsid proteins. Earlier work had already demonstrated that the ITRs were the origins for DNA replication,5Hauswirth WW Berns KI Origin and termination of adeno-associated virus DNA replication.Virology. 1977; 78: 488-499Crossref PubMed Scopus (85) Google Scholar but it was not yet clear what other elements were essential for recombinant virus production, e.g., packaging signals. The first AAV vectors for long-term transduction were constructed by Hermonat and Muzyczka6Hermonat PL Muzyczka N Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells.Proc Natl Acad Sci USA. 1984; 81: 6466-6470Crossref PubMed Scopus (366) Google Scholar and deleted only the capsid-coding region. They demonstrated that they could produce reasonably pure recombinant virus and that the virus could transfer and permanently express a foreign gene in cell culture. A few years later, McLaughlin et al.7McLaughlin SK Collis P Hermonat PL Muzyczka N Adeno-associated virus general transduction vectors: analysis of proviral structures.J Virol. 1988; 62: 1963-1973Crossref PubMed Google Scholar went one step further and showed that all of the AAV coding sequences could be replaced with a transgene cassette that contained appropriate transcriptional control signals. These vectors are the so-called single-stranded AAV vectors that are commonly used today for gene therapy. This work also demonstrated that the only elements needed in cis for vector replication, packaging, and chromosome integration were the ITRs. Several groups then analyzed the integration sites, and Berns and his colleagues discovered that when the AAV rep gene is expressed, AAV genomes preferentially integrate into a unique region of human chromosome 19 (ref. 8Kotin 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 (800) Google Scholar). However, it was also clear that the frequency of transduction was often low in cell culture. The picture changed dramatically in the 1990s. Several groups demonstrated that they could achieve long-term (6–12 months) gene expression following injection into airway, liver, muscle, brain, and eye.9Samulski RJ Muzyczka N AAV-mediated gene therapy for research and therapeutic purposes.Annu Rev Virol. 2014; 1: 427-451Crossref PubMed Scopus (281) Google Scholar This was unheard of at the time. All of the other vector systems appeared to shut off gene expression either through innate or adaptive immune responses or by epigenetic mechanisms. For reasons still not clear, AAV vectors achieved a constant level of expression that persisted. It also became clear that the recombinant genome persisted in animal tissues largely as an episome and had a very low (often undetectable) frequency of integration.10Afione 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,11Nakai H Yant SR Storm TA Fuess S Meuse L Kay MA Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.J Virol. 2001; 75: 6969-6976Crossref PubMed Scopus (373) Google Scholar This meant that the potential for insertional mutagenesis and tumor induction was likely to be low for AAV vectors. The first clinical trial for AAV was reported in 1996. Flotte et al. transduced the cystic fibrosis transmembrane gene (CFTR) into human airway.12Flotte 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.Human Gene Ther. 1996; 7: 1145-1159Crossref PubMed Scopus (219) Google Scholar They demonstrated expression of CFTR and, more importantly, they saw no adverse events. Since then, AAV vectors have been tested in the clinic for a variety of genetic and idiopathic diseases, using both systemic delivery and delivery to specific organs, including eye, brain, liver, muscle, and lung. To date, most of these trials have shown evidence of gene expression and there have been no adverse events attributed to AAV vectors in human trials. Three recent clinical applications are particularly noteworthy. Several groups have shown significant therapeutic efficacy for the correction of RPE65 deficiency, a type of congenital blindness.13Boye SE Boye SL Lewin AS Hauswirth WW A comprehensive review of retinal gene therapy.Mol Ther. 2013; 21: 509-519Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar Nathwani et al.14Nathwani AC Tuddenham EG Rangarajan S Rosales C McIntosh J Linch DC et al.Adenovirus-associated virus vector–mediated gene transfer in hemophilia B.N Engl J Med. 2011; 365: 2357-2365Crossref PubMed Scopus (1354) Google Scholar showed therapeutic efficacy for the correction of factor IX deficiency. And uniQure, a Dutch company, has recently been awarded approval from the European Commission for an AAV-based therapy for lipoprotein lipase deficiency.15Yla-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 (300) Google Scholar This is the first gene therapy drug for a genetic disease that has been approved for human use. These recent developments have captured the attention of the biotech industry, which relies heavily on delivery of drugs that consist of peptides, antibodies, siRNA, or antisense DNA. All of these can be engineered to be expressed long term via AAV, thus obviating the need for repeated drug applications, something that could be particularly useful in undeveloped countries. Thus, AAV gene therapy has the potential to change drug delivery in a fundamental way. It promises not only drugs for single genetic diseases that were previously intractable, but also new strategies for the treatment of diseases of all kinds. And as shown by the work of Gardner et al., it may also solve difficult vaccine problems. A major reason for the current optimism is the simplicity of the vector structure. A nonenveloped, T=1 icosahedron, the viral particle consists of just one protein sequence on its surface repeated 60 times to produce a nanoparticle that is smaller than most synthetic nanoparticles. The X-ray crystal structures of most of the currently used serotypes have been solved (Figure 1), and many laboratories have demonstrated that the capsid can be decorated with novel targeting ligands that improve tissue specificity.16Agbandje-McKenna M Kleinschmidt J AAV capsid structure and cell interactions.Methods Mol Biol. 2011; 807: 47-92Crossref PubMed Scopus (133) Google Scholar Moreover, more than 100 new serotypes of AAV have been isolated from primate tissues.17Gao G Vandenberghe LH Wilson JM New recombinant serotypes of AAV vectors.Curr Gene Ther. 2005; 5: 285-297Crossref PubMed Scopus (413) Google Scholar Understanding the capsid structure has also allowed many groups to create libraries of AAV-containing surface regions that have been shuffled or randomized.18Kotterman MA Schaffer DV Engineering adeno-associated viruses for clinical gene therapy.Nat Rev Genet. 2014; 15: 445-451Crossref PubMed Scopus (520) Google Scholar These in turn can be used to select the best surface properties for targeting a particular tissue by a process called directed evolution. Another major factor in the success of AAV is that it can be made in large quantities and easily purified. A major breakthrough was the development of DNA transfection methods that allowed investigators to make small quantities of pure rAAV that are used today for research purposes.19Xiao X Li J Samulski RJ Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar,20Matsushita T Elliger S Elliger C Podsakoff G Villarreal L Kurtzman GJ et al.Adeno-associated virus vectors can be efficiently produced without helper virus.Gene Ther. 1998; 5: 938-945Crossref PubMed Scopus (414) Google Scholar,21Grimm D Kern A Rittner K Kleinschmidt JA Novel tools for production and purification of recombinant adeno-associated virus vectors.Hum Gene Ther. 1998; 9: 2745-2760Crossref PubMed Scopus (569) Google Scholar In addition, scalable production methods for growing rAAV have been developed, using baculovirus or herpesvirus.22Conway JE Zolotukhin S Muzyczka N Hayward GS Byrne BJ Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.J Virol. 1997; 71: 8780-8789Crossref PubMed Google Scholar,23Smith RH Levy JR Kotin RM A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells.Mol Ther. 2009; 17: 1888-1896Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar These methods are capable of producing ~105 AAV vector genomes per cell, a level achieved by few other mammalian viruses. And since the particle is essentially a protein complex, all of the conventional high-throughput purification methods can be used to manufacture the final clinical product. Moreover, AAV DNA replication has been completed reconstituted in vitro with purified components,24Nash K Chen W Muzyczka N Complete in vitro reconstitution of adeno-associated virus DNA replication requires the minichromosome maintenance complex proteins.J Virol. 2008; 82: 1458-1464Crossref PubMed Scopus (48) Google Scholar and the key genes involved in capsid assembly have been identified.25Sonntag F Schmidt K Kleinschmidt JA A viral assembly factor promotes AAV2 capsid formation in the nucleolus.Proc Natl Acad Sci USA. 2010; 107: 10220-10225Crossref PubMed Scopus (240) Google Scholar It may soon be possible, therefore, to commercially synthesize rAAV in a completely cell-free system. Finally, it is also worth noting that AAV is also creating a quiet revolution in the basic sciences. Because AAV can be used to quantitatively alter the genotype of somatic cells in a permanent way, it can be used to engineer new animal models. The first example of this was by Kirik et al.,26Kirik D Annett LE Burger C Muzyczka N Mandel RJ Bjorklund A Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease.Proc Natl Acad Sci USA. 2003; 100: 2884-2889Crossref PubMed Scopus (340) Google Scholar who overexpressed α-synuclein in the substantia nigra of rats. Their work produced for the first time a model of Parkinson's disease that showed progressive neurodegeneration of dopamine neurons in the brain, something that was not possible using conventional transgenic animal models. This has led more recently to the use of AAV-based expression of channel rhodopsins in the brain to study optogenetics. This approach promises to be an important tool for unraveling the connections in the brain and perhaps telling us how the most sophisticated organ in the body works.27Fenno L Yizhar O Deisseroth K The development and application of optogenetics.Annu Rev Neurosci. 2011; 34: 389-412Crossref PubMed Scopus (1290) Google Scholar By the end of this century, when other types of therapies supplant viral-based gene therapy, the value of AAV as a basic research tool may prove to be its most important contribution.