Adeno-associated virus

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Adeno-associated virus
Adeno-associated virus serotype AAV2.jpg
Adeno-associated virus serotype 2 structure from 1LP3. One fivefold axis shown center.
Virus classification
Group:
Group II (ssDNA)
Order:
Unassigned
Family:
Parvoviridae
Subfamily:
Parvovirinae
Genus:
Dependoparvovirus
Species
  • Adeno-associated dependoparvovirus A
  • Adeno-associated dependoparvovirus B

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Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a very mild immune response, lending further support to its apparent lack of pathogenicity. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur.[1] These features make AAV a very attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models.[2] Recent human clinical trials using AAV for gene therapy in the retina have shown promise.[3]

AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus.

Advantages and drawbacks

Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is 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.[4][5] The 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 (ITR) that aid in concatamer 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.[6][7][8] This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human 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. 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 concatamers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript.

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.[9] Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of cytotoxic T lymphocytes.[10]

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. Accordingly the majority of clinical trials currently underway involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron-specific.

Clinical trials

To date, AAV vectors have been used in over 117 clinical trials worldwide. (Approximately 5.6%)[11] Recently, promising results have been obtained from Phase 1 and Phase 2 trials for a number of diseases, including Leber's Congenital Amaurosis,[12][13][14] Hemophilia,[15] congestive heart failure,[16] lipoprotein lipase deficiency (reference needed), and Parkinson's disease.[17]

Selected Clinical Trials using AAV-Based Vectors[18]
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
Age-Related Macular Degeneration sFlt-1 Subretinal I-II 24 Ongoing
Muscular dystrophy Sarcoglycan Intramuscular I 10 Ongoing
Parkinson's GAD65, GAD67 Intracranial I 12 Complete[19]
Canavan's AAC Intracranial I 21 Ongoing
Batten's CLN2 Intracranial I 10 Ongoing
Alzheimer's NGF Intracranial I 6 Ongoing
Congestive Heart Failure SERCA2a Intra-coronary IIb 250 Ongoing

Trials for the treatment of prostate cancer have reached phase III,[18] however these ex vivo studies do not involve direct administration of AAV to patients.

Pathology

AAV is not considered to have any known role in disease. It has been suggested to have a role in male infertility,[20] as AAV DNA is more commonly found in semen samples from men with abnormal semen. However, no causal link has been found between AAV infection and male infertility.

AAV structure

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.[21]

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.[22] 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,[23][24] as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles.[25]

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.[26][27][28][29]

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.[30] 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 shown to be 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.[24][30][31][32][33][34]

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.[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 that 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]

AAV2 capsid, shown as a ribbon diagram, with the back half hidden for clarity. One fivefold symmetry axis is shown center.

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]

The AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MegaDaltons.[43] The crystal structure of the VP3 protein was determined by Xie, Bue, et al..[44]

AAV serotypes, receptors and native tropism

As of 2006 there have been 11 AAV serotypes described, the 11th in 2004.[45] 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.

Serotype 2

Serotype 2 (AAV2) has been the most extensively examined so far.[46][47][48][49][50][51] AAV2 presents natural tropism towards skeletal muscles,[52] neurons,[46] vascular smooth muscle cells[53] and hepatocytes.[54]

Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (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.[55][56][57] These study results have been disputed by Qiu, Handa, et al..[58] HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.[59]

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,[60] a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania.[61] This could lead to a new anti-cancer agent.

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,[62][63] AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes[64][65][66] and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells.[67] In the brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes.[68] AAV6, a hybrid of AAV1 and AAV2,[66] also shows lower immunogenicity than AAV2.[65]

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),[69] and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.[70]

AAV immunology

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.

Innate

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.[71]

Humoral

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.[72] 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.[73]

Cell-mediated

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.[72] Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring.[74] Combined with data that shows that CD8+ T-cells can recognise elements of the AAV capsid in vitro,[75] 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.[73] 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.[73][74]

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. receptor-mediated endocytosis
  3. endosomal trafficking
  4. escape from the late endosome or lysosome
  5. translocation to the nucleus
  6. uncoating
  7. formation of double-stranded DNA replicative form of the AAV genome
  8. expression of rep genes
  9. genome replication
  10. expression of cap genes, synthesis of progeny ssDNA particles
  11. assembly of complete virions, and
  12. 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.[76]

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,[77][78] by other viruses such as HSV,[79] or by genotoxic agents, such as UV irradiation or hydroxyurea.[80][81][82]

The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al..[77] 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).[83]

See also

References

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