Adeno-associated virus

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Adeno-associated virus
Virus classification
Group: Group II (ssDNA) Family: Parvoviridae Subfamily: Parvovirinae Genus: Dependovirus Species: adeno-associated virus

Adeno-associated virus (AAV) is the smallest of known human viruses. There is no disease which has been to date associated with AAV. The virus causes very mild immune response and can infect non-dividing cells. It incorporates into the host cell's genome, but there is no evidence that it can cause malignant transformation. Because of these features it presents a very attractive subject for creating vectors for gene therapy.

Contents 1 Overview 2 Gene Therapy Vector 3 AAV structure 3.1 AAV genome, transcriptome and proteome 3.1.1 ITR sequences 3.1.2 rep genes and Rep proteins 3.1.3 cap genes and VP proteins 4 AAV serotypes, receptors and native tropism 4.1 Serotype 2 4.1.1 Serotype 2 and cancer 4.2 Other Serotypes 5 AAV infection cycle 6 Notes 7 External links

[edit] Overview

Dependovirus adeno-associated virus belongs to the Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus of mammals (including humans), which has not been associated with any known diseases. AAV is being extensively researched as a potential vector for gene therapy^[1] as it possesses many advantages in this regard.

[edit] Gene Therapy Vector

AAV advantages for gene therapy include: the lack of pathogenicity, the ability to infect non-dividing cells and the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human 19^th chromosome.^[2] The last feature makes it superior to retroviruses, which present 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. AAVs also present very low immunogenicity, restricted only to generation of neutralizing antibodies, while they induce no cytotoxic response.^[3][4][5] This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for the human gene therapy.

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. Other trials have begun, concerning AAV safety for treatment of Canavan disease, muscular dystrophy and late infantile neuronal ceroid lipofuscinosis.

[edit] AAV structure

[edit] AAV genome, transcriptome and proteome

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 inverted terminal repeats (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 together to form a capsid of an icosahedral symmetry.^[6]

[edit] 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 multiplification of the AAV genome.^[7] Another property of these sequences 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 (19^th chromosome in humans) and rescue from it,^[8][9] as well as for efficient encapsidation of the AAV DNA combined with generation of a fully-assembled, deoxyribonuclease-resistant AAV particles.^[10]

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.^{[11][12][13][14]}

[edit] rep genes 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.^[15] 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 showed 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.^{[16][17][15][18][19][9]}

[edit] cap genes and VP proteins

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.^[20] All three of them are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either longer or shorter intron can be excised, which results in formation of two pools of mRNAs: 2.3 kb-, and 2.6 kb-long. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents 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 the ACG sequence, which endcodes threonine, but is surrounded by the optimal Kozak context, that contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.^{[21][22][23][24]}

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.^[25] 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.^[26] Muralidhar et. al. reported that VP2 and VP3 are crucial for correct virion assembly.^[23] 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.^[27]

The crystal structure of the VP3 protein was determined by Xie, Bue, et al.^[28]

[edit] AAV serotypes, receptors and native tropism

As of 2006 there have been 11 AAV serotypes described, the 11^th in 2004.^[29] 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.

[edit] Serotype 2

Serotype 2 (AAV2) has been the most extensively examined so far.^{[30][31][32][33][34][35]} AAV2 presents natural tropism towards e.g. skeletal muscles,^[36] neurons,^[30] vascular smooth muscle cells^[37] and hepatocytes.^[38]

Three cell receptors haver been described for AAV2: heparan sulfate proteoglican (HSPG), a_Vß₅ 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.^[39][40][41]) These study results have been disputed by Qiu, Handa, et al.^[42] HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.^[43]

[edit] Serotype 2 and cancer

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, a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania.^[44] This could lead to a new anti-cancer agent.

[edit] Other Serotypes

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^[45][46][47] and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells.^[48] AAV6, a hybrid of AAV1 and AAV2,^[47] also shows lower immunogenicity than AAV2.^[46]

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),^[49] and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.^[50]

[edit] AAV infection cycle

There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles:

1. attachment to the cell membrane
2. endocytosis
3. endosomal trafficking
4. escape from the late endosome or lysosome
5. translocation to the nucleus
6. formation of double-stranded DNA replicative form of the AAV genome
7. rep genes expression
8. genome replication
9. cap genes expression, synthesis of progeny ssDNA particles
10. assembly of complete virions, and
11. release from the infected cell.

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.^[51]

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,^[52][53] by other viruses such as HSV,^[54] or by genotoxic agents, such as UV irradiation or hydroxyurea.^[55][56][57]

The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al.^[52] 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).^[58]

[edit] Notes

[edit] External links

• http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GeneTherapy2.html