adeno-associated virus adeno-associated virus adeno-associated virus adeno-associated virus serotype 2 structure from 1lp3. one fivefold axis shown center. scientific classification (unranked): virus realm: monodnaviria kingdom: shotokuvirae phylum: cossaviricota class: quintoviricetes order: piccovirales family: parvoviridae subfamily: parvovirinae genus: dependoparvovirus viruses included: adeno-associated dependoparvovirus a adeno-associated dependoparvovirus b adeno-associated viruses (aav) are small viruses that infect humans and some other primate species. they belong to the genus dependoparvovirus, which in turn belongs to the family parvoviridae. they are small (approximately 26 nm in diameter) replication-defective, nonenveloped viruses and have linear single-stranded dna (ssdna) genome of approximately 4.8 kilobases (kb). several features make aav an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. gene therapy vectors using aav can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. in the native virus, however, integration of virally carried genes into the host genome does occur. integration can be important for certain applications, but can also have unwanted consequences. recent human clinical trials using aav for gene therapy in the retina have shown promise. in march 2023, a series of nature papers linked infection of adeno-associated virus 2 (aav2) to a wave of childhood hepatitis. history the adeno-associated virus (aav), previously thought to be a contaminant in adenovirus preparations, was first identified as a dependoparvovirus in the 1960s in the laboratories of bob atchison at pittsburgh and wallace rowe at nih. serological studies in humans subsequently indicated that, despite being present in people infected by helper viruses such as adenovirus or herpes virus, aav itself did not cause any disease. use in gene therapy advantages and drawbacks wild-type aav has attracted considerable interest from gene therapy researchers due to a number of features. chief amongst these was the virus's apparent lack of pathogenicity. it can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated aavs1) in the human chromosome 19. this feature makes it somewhat more predictable than retroviruses, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. the aav genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. development of aavs as gene therapy vectors, however, has eliminated this integrative capacity by removal of the rep and cap from the dna of the vector. the desired gene together with a promoter to drive transcription of the gene is inserted between the inverted terminal repeats (itrs) that aid in concatemer formation in the nucleus after the single-stranded vector dna is converted by host cell dna polymerase complexes into double-stranded dna. aav-based gene therapy vectors form episomal concatemers in the host cell nucleus. in non-dividing cells, these concatemers remain intact for the life of the host cell. in dividing cells, aav dna is lost through cell division, since the episomal dna is not replicated along with the host cell dna. random integration of aav dna into the host genome is detectable but occurs at very low frequency. aavs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response. this feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human gene therapy. use of the virus does present some disadvantages. the cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. large genes are, therefore, not suitable for use in a standard aav vector. options are currently being explored to overcome the limited coding capacity. the aav itrs of two genomes can anneal to form head-to-tail concatemers, almost doubling the capacity of the vector. insertion of splice sites allows for the removal of the itrs from the transcript. because of aav's specialized gene therapy advantages, researchers have created an altered version of aav termed self-complementary adeno-associated virus (scaav). whereas aav packages a single strand of dna and must wait for its second strand to be synthesized, scaav packages two shorter strands that are complementary to each other. by avoiding second-strand synthesis, scaav can express more quickly, although as a caveat, scaav can only encode half of the already limited capacity of aav. recent reports suggest that scaav vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic t lymphocytes. humoral immunity instigated by infection with the wild type is thought to be common. the associated neutralising activity limits the usefulness of the most commonly used serotype aav2 in certain applications. accordingly, the majority of clinical trials under way involve delivery of aav2 into the brain, a relatively immunologically privileged organ. in the brain, aav2 is strongly neuron-specific. clinical trials as of 2019, aav vectors have been used in over 250 clinical trials worldwide, approximately 8.3% of virus-vectored gene-therapy trials. recently, promising results have been obtained from phase 1 and phase 2 trials for a number of diseases, including leber's congenital amaurosis, hemophilia, congestive heart failure, spinal muscular atrophy, lipoprotein lipase deficiency, and parkinson's disease. selected clinical trials using aav-based vectors indicationgeneroute of administrationphasesubject numberstatus cystic fibrosiscftrlung, via aerosoli12complete cftrlung, via aerosolii38complete cftrlung, via aerosolii100complete hemophilia bfixintramusculari9complete fixhepatic arteryi6ended arthritistnfr:fcintraarticulari1ongoing hereditary emphysemaaatintramusculari12ongoing leber's congenital amaurosisrpe65subretinali–iimultipleseveral ongoing and complete (voretigene neparvovec) age-related macular degenerationsflt-1subretinali–ii24ongoing duchenne muscular dystrophysgcaintramusculari10ongoing parkinson's diseasegad65, gad67intracraniali12complete canavan diseaseaacintracraniali21ongoing batten diseasecln2intracraniali10ongoing alzheimer's diseasengfintracraniali6ongoing spinal muscular atrophysmn1intravenous, intrathecali–iii150+several ongoing and complete (onasemnogene abeparvovec) congestive heart failureserca2aintra-coronaryiib250ongoing fundamental biology two adenovirus particles surrounded by numerous, smaller adeno-associated viruses (negative-staining electron microscopy, magnification approximately 200,000×) genomics, transcriptomics and proteomics the aav genome is built of single-stranded deoxyribonucleic acid (ssdna), either positive- or negative-sensed, which is about 4.7 kilobase long. the genome comprises itrs at both ends of the dna strand, and two open reading frames (orfs): rep and cap. the former is composed of four overlapping genes encoding rep proteins required for the aav life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: vp1, vp2 and vp3, which interact to form a capsid with icosahedral symmetry. itr sequences the inverted terminal repeat (itr) sequences comprise 145 bases each. they were named so because of their symmetry, which was shown to be required for efficient multiplication of the aav genome. the feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second dna strand. the itrs were also shown to be required for both integration of the aav dna into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the aav dna combined with generation of a fully assembled, deoxyribonuclease-resistant aav particles. with regard to gene therapy, itrs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. with this assumption many methods were established for efficient production of recombinant aav (raav) vectors containing a reporter or therapeutic gene. however, it was also published that the itrs are not the only elements required in cis for the effective replication and encapsidation. a few research groups have identified a sequence designated cis-acting rep-dependent element (care) inside the coding sequence of the rep gene. care was shown to augment the replication and encapsidation when present in cis. rep gene and rep proteins on the "left side" of the genome there are two promoters called p5 and p19, from which two overlapping messenger ribonucleic acids (mrnas) of different length can be produced. each of these contains an intron which can be either spliced out or not. given these possibilities, four various mrnas, and consequently four various rep proteins with overlapping sequence can be synthesized. their names depict their sizes in kilodaltons (kda): rep78, rep68, rep52 and rep40. rep78 and 68 can specifically bind the hairpin formed by the itr in the self-priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. they were also shown to be necessary for the aavs1-specific integration of the aav genome. all four rep proteins were shown to bind atp and to possess helicase activity. it was also shown that they upregulate the transcription from the p40 promoter (mentioned below), but downregulate both p5 and p19 promoters. cap gene and vp proteins the right side of a positive-sensed aav genome encodes overlapping sequences of three capsid proteins, vp1, vp2 and vp3, and two accessory proteins, maap & aap, which start from one promoter, designated p40. the molecular weights of these proteins are 87, 72 and 62 kilodaltons, respectively. the aav capsid is composed of a mixture of vp1, vp2, and vp3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1:1:10, with an empty mass of approximately 3.8 mda. the crystal structure of the vp3 protein was determined by xie, bue, et al. aav2 capsid, shown as a ribbon diagram, with the back half hidden for clarity. one fivefold symmetry axis is shown center. the cap gene produces an additional, non-structural protein called the assembly-activating protein (aap). this protein is produced from orf2 and is essential for the capsid-assembly process. the exact function of this protein in the assembly process and its structure have not been solved to date. all three vps are translated from one mrna. after this mrna is synthesized, it can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two pools of mrnas: a 2.3 kb- and a 2.6 kb-long mrna pool. usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mrna represents the so-called "major splice". in this form the first aug codon, from which the synthesis of vp1 protein starts, is cut out, resulting in a reduced overall level of vp1 protein synthesis. the first aug codon that remains in the major splice is the initiation codon for vp3 protein. however, upstream of that codon in the same open reading frame lies an acg sequence (encoding threonine) which is surrounded by an optimal kozak context. this contributes to a low level of synthesis of vp2 protein, which is actually vp3 protein with additional n terminal residues, as is vp1. since the bigger intron is preferred to be spliced out, and since in the major splice the acg codon is a much weaker translation initiation signal, the ratio at which the aav structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle. the unique fragment at the n terminus of vp1 protein was shown to possess the phospholipase a2 (pla2) activity, which is probably required for the releasing of aav particles from late endosomes. muralidhar et al. reported that vp2 and vp3 are crucial for correct virion assembly. more recently, however, warrington et al. showed vp2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that vp2 can tolerate large insertions in its n terminus, while vp1 can not, probably because of the pla2 domain presence. post-translational modifications recent discoveries made through use of high-throughput 'omics approaches include the fact that aav capsids are post-translationally modified (ptm) during production such as acetylation, methylation, phosphorylation, deamidation, o-glycnacylation and sumoylation throughout capsid proteins vp1, vp2 and vp3. these ptms differ depending on the manufacturing production platform. another such discovery is the fact that aav genomes are epigenetically methylated during production. besides price, these findings might affect expression kinetics, raav receptor binding, trafficking, vector immunogenicity, and expression durability. classification, serotypes, receptors and native tropism in humans serotypetissue tropismpass the blood brain barriercell tropismsyntheticcommentpublished cap / rep 2smooth muscle, cns, livernono 5cns, smooth musclenono 8cns, brain, liver, smooth musclenono 9cns, liver, smooth muscleyesno pal2cnsyesyesliver expression is 1/4 of aav9no 9p1/aavmyocnsyesastrocytesyeshigh musculature including skeletal muscle, heart and diaphragmsckeletal transductionno two species of aav were recognised by the international committee on taxonomy of viruses in 2013: adeno-associated dependoparvovirus a (formerly aav-1, −2, −3 and −4) and adeno-associated dependoparvovirus b (formerly aav-5). until the 1990s, virtually all aav biology was studied using