The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively.[35] All three of them 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, which 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.[36][37][38][39]
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.[40] 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.[41] Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly.[38] 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.[42]
Three cell receptors have been described for AAV2: heparan sulfate proteoglican (HSPG), aVβ5integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis.[54][55][56]) These study results have been disputed by Qiu, Handa, et al..[57] HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.[58]
Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers,[59] a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania.[60] This could lead to a new anti-cancer agent.
他の血清型
Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes[61][62][63] and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells.[64] AAV6, a hybrid of AAV1 and AAV2,[63] also shows lower immunogenicity than AAV2.[62]
Serotypes can differ with the respect to the receptors they are bound to. For example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes),[65] and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.[66]
The innate immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into the liver, which seems to sequester a large percentage of the injected viral particles. Both soluble factor levels and cell infiltration appear to return to baseline within six hours. By contrast, more aggressive viruses produce innate responses lasting 24 hours or longer.[67]
液性免疫
The virus is known to instigate robust humoral immunity in animal models and in the human population where up to 80% of individuals are thought to be seropositive for AAV2. Antibodies are known to be neutralising and for gene therapy applications these do impact on vector transduction efficiency via some routes of administration. As well as persistent AAV specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that the B-cell memory is also strong.[68] In seropositive humans, circulating IgG antibodies for AAV2 appear to be primarily composed of the IgG1 and IgG2 subclasses, with little or no IgG3 or IgG4 present.[69]
細胞免疫
The cell-mediated response to the virus and to vectors is poorly characterised and has been largely ignored in the literature as recently as 2005.[68] Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring.[70] Combined with data that shows that CD8+ T-cells can recognise elements of the AAV capsid in vitro[71], it appears that there may be a cytotoxic T lymphocyte response to AAV vectors. Cytotoxic responses would imply the involvement of CD4+ T helper cells in the response to AAV and in vitro data from human studies suggests that the virus may indeed induce such responses including both Th1 and Th2 memory responses.[69] A number of candidate T cell stimulating epitopes have been identified within the AAV capsid protein VP1, which may be attractive targets for modification of the capsid if the virus is to be used as a vector for gene therapy.[69][70]
Some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of AAV. Replication of the virus can also vary in one cell type, depending on the cell's current cell cycle phase.[71] The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. The first factor that was described as providing successful generation of new AAV particles, was the adenovirus, from which the AAV name originated. It was then shown that AAV replication can be facilitated by selected proteins derived from the adenovirus genome,[72][73] by other viruses such as HSV,[74] or by genotoxic agents, such as UV irradiation or hydroxyurea.[75][76][77]
The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al..[72] This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. In the absence of helper virus or genotoxic factors, AAV DNA can either integrate into the host genome or persist in episomal form. In the former case integration is mediated by Rep78 and Rep68 proteins and requires the presence of ITRs flanking the region being integrated. In mice, the AAV genome has been observed persisting for long periods of time in quiescent tissues, such as skeletal muscles, in episomal form (a circular head-to-tail conformation).[78]
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