AAV2 was first discovered in an Adenovirus type 12 preparation as contaminant in 1965 144. AAV1, AAV3, and AAV4 were sequentially isolated from a simian Adenovirus 15 stock, an Adenovirus 7 stock, and African green monkey 145-147. AAV5 and AAV9 were discovered from humans, while AAV7 and AAV8 were isolated from rhesus monkeys 124,148. In addition, AAV10 and AAV11 were cloned from cynomolgus monkeys 149. In 2004, a study led by Gao G. expand the collection of naturally occurring AAV to more than 100 members 124. The pursuit of more naturally occurring AAV strains and reengineered variants is fueled by AAV’s encouraging successes as gene therapy vectors.
The idea of using AAV to package transgene traces back to 1982 150,151. The era of gene therapy flourishes when therapeutic genes can be introduced to target cells or organs. Compared to other viral vectors, AAV has the advantage of no currently-known pathogenicity, prolonged transgene expression kinetics, and the ability to transduce both dividing and non-dividing cells. Transition of AAV vectors from bench-to-bedside was enabled by the extensive knowledge of the basic virology of the vectors described earlier.
Preclinical and clinical trials have been carried out with many AAV strains and emerging AAV variants 152. For instance, AAV8 has been tested in nonhuman primates and dog models to express factor IX in liver, with the hope of alleviating hemophilia symptoms. AAV1 packaging the SERCA2a gene is in phase I/II clinical trial to treat cardiac failure. As described earlier, AAV5, HAE-1/2, and AAV2.5T have been investigate to develop cures for pulmonary diseases, such as cystic fibrosis. In the past few years, extremely exciting results of recombinant AAV vectors come from clinical trials of Leber’s congenital amaurosis. AAV4 and AAV8 restoring retinal pigmented epithelium (RPE) functions showed success in
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expression RPE65 gene into RPE65-deficient dogs and nonhuman primate models. Patient treatments demonstrated over five orders of magnitude of progression in rod photoreceptor function. Using AAV as delivery machinery into the brain also saw encouraging results in treating Canavan’s, Parkinson’s, and Batten’s diseases in long-term assessments.
Despite persistent successes in the clinical front, there are still concerns and challenges regarding to the pre-existing humoral immunity, cross-species difference in tropism, immune response to both AAV vector cassette and transgene products. Further improvement of existing vectors and development of new AAV vectors counts on a persistent accumulation of basic knowledge about AAV biology.
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Table 1. Examples of Viruses and Their Cognate Glycan Receptors1
Virus Family Virus Type Glycan Receptor
Adenoviridae Adeno 37 Adenovirus 2,5
α2-3 SA
Heparan Sulfate
Arenaviridae Lassa Virus Dystroglycan Glycans
Caliciviridae
Noroviruses Norwalk and others Histo-blood group Coronaviridae Coronavirus OC43 9-O-acetyl SA Flaviviridae
Hepaciviruses Flavivirus
Hepatitis C Dengue Virus
Japanese encephalitis Virus West Nile Virus
Heparan Sulfate Heparan Sulfate Heparan Sulfate Herpesviridae α-herpesviruses β-herpesviruses γ-herpesviruses
Herpes simplex virus types 1 & 2 Varicella-zoster virus
Cytomegalovirus
Human Herpesvirus Types 6 & 7 Human Herpesvirus type 8
Heparan Sulfate Heparan Sulfate Heparan Sulfate Heparan Sulfate Ortomyxoviridae Influenza A virus
Influenza B virus Influenza C virus α2-3 SA, α2-6 SA α2-3 SA, α2-6 SA 9-O-acetylsialic acid Papillomaviridae Papillomavirus Human papillomavirus types 11, 16, 33 Heparan sulfate Paramyxoviridae Respirovurus Pneumovirus Metapneumov. Paramyxovirus 1-3
Respiratory syncytial virus Human metapneumovirus Sialic acid Heparan sulfate (chondroitin sulfate) Heparan sulfate Parvoviridae Erythrovirus Dependovirus (see section 1.2) B19
Adeno associated virus (AAV) types 4 & 5 AAV type 2 Globoside/Histo-blood group P substance SA Heparan sulfate Picornavirus Enterovirus Rhinovirus Enterovirus 70 Rhinovirus 87 SA SA Polyomaviridae
polyomavirus JC and BK virus SA Poxviridae
Ortopoxvirus Reoviridae
Vaccinia virus Heparan Sulfate
(Chondroitin Sulfate)
Ortoreovirus Reovirus 3 SA
Rotavirus Rotavitus SA
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Table 2 Structure, Receptor, and Tropism of AAV1-9
AAV Serotype
VP Structure
(PDB#)
Glycan Receptor Protein
Receptor Tropism
1 3NG9 N-linked SA Muscle, Lung
2 1LP3 Heparan Sulfate αVβ5 & α5β1 Integrins, HGFR, FGFR,
LamR
Liver, Neuron
3B 3KIE Heparan Sulfate LamR Liver
4 2G8G O-linked SA Heart, Lung,
Astrocytes 5 3NTT N-linked SA PDGFR Muscle 6 3OAH N-linked SA Heparan Sulfate EGFR Muscle 7 Muscle, Liver 8 2QA0 Liver
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Figure 1. Genomic Map of AAV. There are two ORFs between ITRs, coding non-structural and structural proteins of AAV. The p5 promoter initiates transcription of Rep78 and Rep68, while p19 drives that of Rep52 and Rep48. Transcripts of non-structural protein, AAP, and structural proteins, VP1, 2, and 3, are driven by p40 promoter. Alternative splicing results in mRNAs coding for VP1 and VP2. Leaky scanning of the stop codon in the middle of VP2 yields VP3.
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Figure 2. Structural Components of AAV Capsid. (A) Schematic diagram of the eight- strand jelly-roll β-barrel structure displayed by many icosahedral viral capsids. Adapted from reference 153. Eight β strands are sequentially numbered B-I. Loops connecting the neighboring two strands are named by numbers of the two connecting strands. (B) Ribbon rendering of AAV2 VP3 (PDB# 1LP3) showing β strands corresponding to (A) in blue. The conserved short α-helix is colored in red. DE loop and HI loop forming the five-fold axes of symmetry, and GH loop on the three-fold axes are labeled. Two-, three-, five-fold axes of symmetry are marked by oval, triangle, and pentagon, respectively to orient the view. (C) A trimer model of AAV2 VP3, with individual chain colored in palecyan, bluewhite, and lightorange in PyMOL. (D) A pentamer model of AAV2 VP3, showing the five-fold axes of symmetry. Each VP monomer is colored wheat, palegreen, lightblue, paleyellow, and light pink, respectively. (E) Visualization of a full capsid model of AAV2, looking into the five- fold axes of symmetry.