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 in diameter) replication-defective, non-enveloped viruses and have linear single-stranded DNA (ssDNA) genomes of approximately 4.8 kilobases (kb).
Several features make AAV an attractive candidate for creating viral vectors in the field of molecular biology including animal modeling and the creation of isogenic human disease models. Vectors using AAV can infect both dividing and quiescent cells and persist in an non-integrated extrachromosomal state into the genome of the host cell. Low level integration into the host genome occurs, particularly in the wild type virus setting.
Recombinant or replication-defective AAV has emerged an important technology in the field of gene therapy. This has now led to multiple approved drugs leveraging its gene delivery often for genetic disorders such as Spinal Muscular Atrophy, forms of blindness, and hemophilia. The use of AAV has its limitations including the limited genetic cargo capacity and adverse events, particularly at very high dose levels have been recorded.
In March 2023, a series of Nature papers detected high titres of adeno-associated virus 2 (AAV2), alongside adenovirus and herpesvirus, in samples from a wave of childhood hepatitis. One paper suggested that AAV2 co-infection may contribute to more serious liver disease than infection with only adeno- or herpesviruses and that the causal link remains to be established.
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 University of Pittsburgh and Wallace Rowe at the NIH. Serological studies in humans subsequently indicated that, despite being present in people infected by helper viruses such as adenovirus or herpes virus, AAV was thought to be not associated with disease. At present, adeno-associated virus (AAV) vectors are considered the gold standard in gene therapy.
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.
The primary method for AAV production involves transient transfection of HEK-293 cells using three plasmids that carry the therapeutic gene, AAV rep/cap genes, and helper adenoviral genes. The production process includes cultivation, particularly in bioreactors, and purification aimed at increasing the yield of full capsids and removing impurities. To improve productivity, new methods are being developed, such as using engineered recombinant helper viruses and establishing stable producer cell lines.
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. CRISPR allows the use of sequential insertion at any desired site. The first vector recognizes the target locus and inserts half of the desired gene. The second vector then recognizes the half-done insertion and inserts the other half.
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 Indication Gene Route of administration Phase Subject number Status Cystic fibrosis CFTR Lung, via aerosol I 12 Complete CFTR Lung, via aerosol II 38 Complete CFTR Lung, via aerosol II 100 Complete Hemophilia B FIX Intramuscular I 9 Complete FIX Hepatic artery I 6 Ended Arthritis TNFR:Fc Intraarticular I 1 Ongoing Hereditary emphysema AAT Intramuscular I 12 Ongoing Leber's congenital amaurosis RPE65 Subretinal I–II Multiple Several ongoing and complete (voretigene neparvovec) Age-related macular degeneration sFlt-1 Subretinal I–II 24 Ongoing Duchenne muscular dystrophy SGCA Intramuscular I 10 Ongoing Parkinson's disease GAD65, GAD67 Intracranial I 12 Complete Canavan disease AAC Intracranial I 21 Ongoing Batten disease CLN2 Intracranial I 10 Ongoing Alzheimer's disease NGF Intracranial I 6 Ongoing Spinal muscular atrophy SMN1 Intravenous, intrathecal I–III 150+ Several ongoing and complete (onasemnogene abeparvovec) Congestive heart failure SERCA2a Intra-coronary IIb 250 Ongoing
FDA Approved AAV Gene Therapy Products AAV based gene therapy products have entered the worldwide market following completion of successful clinical trials. The table below contains FDA approved gene therapies that have entered the American Market and possibly other markets. +AAV Based FDA-Approved Gene Therapy Drug NameIndicationDate of FDA Approval AnnouncementCompany MarketingLUXTURNARPE65 Mutation-associated retinal dystrophy19 December 2017Spark TherapeutcsHEMGENIXHemophilia B 22 November 2022CSL BehringROCTAVIANHemophilia A30 June 2023BioMarin PharmaceuticalBEQVEZHemophilia B25 April 2024PfizerELEVIDYSDuchenne Muscular Dystrophy (DMD)Either 20 June or 29 August 2024Sarepta TherapeuticsZOLGENSMASpinal Muscular Atrophy (SMA)26 July 2024Novartis Gene TherapiesKEBILIDIAromatic L Amino-Acid Decarboxylase (AADC) Deficiency11 November 2024PTC TherapeuticsITVISMASpinal Muscular Atrophy (SMA)24 November 2025Novartis Gene TherapiesOTARMENIBiallelic OTOF mutation associated Sensorineural hearing loss23 April 2026Regeneron PharmaceuticalsGENGLYCOSGlycogen Storage Disease Type Ia19 August 2026UltragenyxFAYUVISanfilippo Syndrome Type A17 September 2026Ultragenyx
Fundamental biology
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