Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for 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. It is accordingly unclear if the site-specific integration can be preserved in a usable vector since it appears to be partially dependent on products of the Rep open reading frame. The humoral immunity instigated by infection with the wild type is thought to be a very common event. The associated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications.
To date, AAV vectors have been used for first- and second-phase clinical trials for treatment of cystic fibrosis and first-phase trials for hemophilia. Promising results have been obtained from phase I trials for Parkinson's disease, showing good tolerance of an AAV2 vector in the central nervous system. Other trials have begun, concerning AAV safety for treatment of Canavan disease, muscular dystrophy and late infantile neuronal ceroid lipofuscinosis.
|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|
Trials for the treatment of prostate cancer have reached phase III, however these ex vivo studies do not involve direct administration of AAV to patients.
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) genes 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.
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.
The crystal structure of the VP3 protein was determined by Xie, Bue, et al.
As of 2006 there have been 11 AAV serotypes described, the 11th in 2004. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy.
Three cell receptors have been described for AAV2: heparan sulfate proteoglican (HSPG), aVβ5 integrin 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.) These study results have been disputed by Qiu, Handa, et al. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.
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 and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.
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), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.
AAV is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology.
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 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.
There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles:
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.
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, by other viruses such as HSV, or by genotoxic agents, such as UV irradiation or hydroxyurea.
The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al. 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).