Adeno-associated virus
Encyclopedia
Adeno-associated virus (AAV) is a small virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

 which infects humans and some other primate species. AAV is not currently known to cause disease
Disease
A disease is an abnormal condition affecting the body of an organism. It is often construed to be a medical condition associated with specific symptoms and signs. It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune...

 and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....

 into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...

, and for the creation of isogenic human disease models
Isogenic human disease models
Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...

. Recent human clinical trials using AAV for gene therapy in the retina
Adeno associated virus and gene therapy of the human retina
Gene therapy is the use of genetic material inserted into a patient’s cell for the treatment of an inherited or acquired diseases. There are many medical conditions that are a result of mutation in patient’s gene...

 have shown promise.

AAV belongs to the genus
Genus
In biology, a genus is a low-level taxonomic rank used in the biological classification of living and fossil organisms, which is an example of definition by genus and differentia...

 Dependovirus
Dependovirus
Dependovirus is a genus of the Parvoviridae viruses, which are Group II viruses according to the Baltimore classification. The Dependovirus is part of the sub family of the Parvoviridae family known as the Parvovirinae...

, which in turn belongs to the family
Family (biology)
In biological classification, family is* a taxonomic rank. Other well-known ranks are life, domain, kingdom, phylum, class, order, genus, and species, with family fitting between order and genus. As for the other well-known ranks, there is the option of an immediately lower rank, indicated by the...

 Parvoviridae
Parvoviridae
The Parvoviridae family includes the smallest known viruses, and some of the most environmentally resistant. They were discovered during the 1960s and affect vertebrates and insects...

. The virus is a small (20 nm
Nanometre
A nanometre is a unit of length in the metric system, equal to one billionth of a metre. The name combines the SI prefix nano- with the parent unit name metre .The nanometre is often used to express dimensions on the atomic scale: the diameter...

) 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 viruses' 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
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 19. The feature makes it somewhat more predictable than retrovirus
Retrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...

es, which present the threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. Development of AAV's 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 concatamers in the host cell nucleus. In non-dividing cells, these concatamers 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 low but detectable. AAV's also present very low immunogenicity
Immunogenicity
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human or animal.- Immunogenicity :The ability to induce humoral and/or cell-mediated immune responses....

, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly defined cytotoxic response
Antibody-dependent cellular cytotoxicity
Antibody-Dependent Cell-Mediated Cytotoxicity is a mechanism of cell-mediated immunity whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune...

. This feature, along with the ability to infect quiescent cells
G0 phase
The G0 phase is a period in the cell cycle in which cells exist in a quiescent state. G0 phase is viewed as either an extended G1 phase, where the cell is neither dividing nor preparing to divide, or a distinct quiescent stage that occurs outside of the cell cycle...

 present their dominance over adenoviruses as vectors for the human gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...

.

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.

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 for first- and second-phase clinical trials for treatment of cystic fibrosis
Cystic fibrosis
Cystic fibrosis is a recessive genetic disease affecting most critically the lungs, and also the pancreas, liver, and intestine...

 and first-phase trials for hemophilia. Promising results have been obtained from phase I trials for Parkinson's disease
Parkinson's disease
Parkinson's disease is a degenerative disorder of the central nervous system...

, showing good tolerance of an AAV2 vector in the central nervous system. Other trials have begun, concerning AAV safety for treatment of Canavan disease
Canavan disease
Canavan disease, also called Canavan-Van Bogaert-Bertrand disease, aspartoacylase deficiency or aminoacylase 2 deficiency, is an autosomal recessive degenerative disorder that causes progressive damage to nerve cells in the brain. Canavan disease is also one of the most common degenerative...

, muscular dystrophy
Muscular dystrophy
Muscular dystrophy is a group of muscle diseases that weaken the musculoskeletal system and hamper locomotion. Muscular dystrophies are characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue.In the 1860s, descriptions of boys who...

 and late infantile neuronal ceroid lipofuscinosis
Neuronal Ceroid Lipofuscinosis
Neuronal Ceroid Lipofuscinoses is the general name for a family of at least eight genetically separate neurodegenerative disorders that result from excessive accumulation of lipopigments in the body's tissues. These lipopigments are made up of fats and proteins...

.
Selection of Clinical Trials using AAV-Based Vectors
Indication Gene Route of administration Phase Subject number Status
Cystic fibrosis CFTR
Cystic fibrosis transmembrane conductance regulator
Cystic fibrosis transmembrane conductance regulator is a protein that in humans is encoded by the CFTR gene.CFTR is a ABC transporter-class ion channel that transports chloride and thiocyanate ions across epithelial cell membranes...

Lung, via aerosol I 12 Complete
CFTR Lung, via aerosol II 38 Complete
CFTR Lung, via aerosol II 100 Complete
Hemophilia B FIX
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...

Intramuscular I 9 Complete
FIX Hepatic artery I 6 Ended
Arthritis
Arthritis
Arthritis is a form of joint disorder that involves inflammation of one or more joints....

TNFR:Fc Intraarticular I 1 Ongoing
Hereditary emphysema AAT Intramuscular I 12 Ongoing
Muscular dystrophy Sarcoglycan Intramuscular I 10 Ongoing
Parkinson's GAD65, GAD67 Intracranial I 12 Complete
Canavan's AAC Intracranial I 21 Ongoing
Batten's CLN2 Intracranial I 10 Ongoing
Alzheimer's NGF Intracranial I 6 Ongoing


Trials for the treatment of prostate cancer
Prostate cancer
Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize from the prostate to other parts of the body, particularly...

 have reached phase III, 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
Infertility
Infertility primarily refers to the biological inability of a person to contribute to conception. Infertility may also refer to the state of a woman who is unable to carry a pregnancy to full term...

, 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 genome, transcriptome and proteome

The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

), 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 frame
Open reading frame
In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...

s (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
Capsid
A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic...

 proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.

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. Another property of these sequences is their ability to form a hairpin
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...

, which contributes to so-called self-priming that allows primase
Primase
DNA primase is an enzyme involved in the replication of DNA.Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template...

-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, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease
Deoxyribonuclease
A deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Thus, deoxyribonucleases are one type of nuclease...

-resistant AAV particles.

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
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...

 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.

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
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...

 which can be either spliced
Splicing (genetics)
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...

 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
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...

 (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind the hairpin
Hairpin
A hair pin or hairpin is a long device used to hold a person's hair in place.Hairpins made of metal, ivory, bronze, carved wood, etc. were used in ancient Assyria and Egypt for securing decorated hairstyles. Such hairpins suggest, as graves show, that many were luxury objects among the Egyptians...

 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
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...

 and to possess helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...

 activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below), but downregulate both p5 and p19 promoters.

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. All three of them are translated from one mRNA. After this mRNA is synthesized, it can be spliced
Splicing (genetics)
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...

 in two different manners: either a longer or shorter intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...

 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
Start codon
The start codon is generally defined as the point, sequence, at which a ribosome begins to translate a sequence of RNA into amino acids.When an RNA transcript is "read" from the 5' carbon to the 3' carbon by the ribosome the start codon is the first codon on which the tRNA bound to Met,...

, 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
Kozak consensus sequence
The Kozak consensus sequence, Kozak consensus or Kozak sequence, is a sequence which occurs on eukaryotic mRNA and has the consensus gccRccAUGG, where R is a purine three bases upstream of the start codon , which is followed by another 'G'. The Kozak consensus sequence plays a major role in the...

. This contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.

Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...

 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
Phospholipase
A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acids and other lipophilic substances. There are four major classes, termed A, B, C and D, distinguished by the type of reaction which they catalyze:*Phospholipase A...

 A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosome
Endosome
In biology, an endosome is a membrane-bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for...

s. 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 AAV capsid is composed of 60 capsid protein subunits, VP1, VP2, and VP3, that are arranged in a icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3,900 KiloDaltons.
The crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...

 of the VP3 protein was determined by Xie, Bue, et al..

AAV serotypes, receptors and native tropism

As of 2006 there have been 11 AAV serotype
Serotype
Serotype or serovar refers to distinct variations within a subspecies of bacteria or viruses. These microorganisms, viruses, or cells are classified together based on their cell surface antigens...

s 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.

Serotype 2

Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards skeletal muscle
Skeletal muscle
Skeletal muscle is a form of striated muscle tissue existing under control of the somatic nervous system- i.e. it is voluntarily controlled. It is one of three major muscle types, the others being cardiac and smooth muscle...

s, neuron
Neuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...

s, vascular smooth muscle
Vascular smooth muscle
Vascular smooth muscle refers to the particular type of smooth muscle found within, and composing the majority of the wall of blood vessels.Vascular smooth muscle contracts or relaxes to both change the volume of blood vessels and the local blood pressure, a mechanism that is responsible for the...

 cells and hepatocyte
Hepatocyte
A hepatocyte is a cell of the main tissue of the liver. Hepatocytes make up 70-80% of the liver's cytoplasmic mass.These cells are involved in:* Protein synthesis* Protein storage* Transformation of carbohydrates...

s.

Three cell receptors have been described for AAV2: heparan sulfate proteoglycan (HSPG), aVβ5 integrin
Integrin
Integrins are receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the ECM. They also play a role in cell signaling and thereby regulate cellular shape, motility, and the cell cycle....

 and fibroblast growth factor
Fibroblast growth factor
Fibroblast growth factors, or FGFs, are a family of growth factors involved in angiogenesis, wound healing, and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal...

 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
Endocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...

.) These study results have been disputed by Qiu, Handa, et al.. HSPG functions as the primary receptor, though its abundance in the extracellular matrix
Extracellular matrix
In biology, the extracellular matrix is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals.Extracellular...

 can scavenge AAV particles and impair the infection efficiency.

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
Immunology
Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders ; the...

 and microbiology
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

 at the Penn State College of Medicine in Pennsylvania
Pennsylvania
The Commonwealth of Pennsylvania is a U.S. state that is located in the Northeastern and Mid-Atlantic regions of the United States. The state borders Delaware and Maryland to the south, West Virginia to the southwest, Ohio to the west, New York and Ontario, Canada, to the north, and New Jersey to...

. 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, 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 acid
Sialic acid
Sialic acid is a generic term for the N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. It is also the name for the most common member of this group, N-acetylneuraminic acid...

s (of different form for each of these serotypes), and AAV5 was shown to enter cells via the platelet-derived growth factor
Platelet-derived growth factor
In molecular biology, platelet-derived growth factor is one of the numerous growth factors, or proteins that regulate cell growth and division. In particular, it plays a significant role in blood vessel formation , the growth of blood vessels from already-existing blood vessel tissue. Uncontrolled...

 receptor.

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
Pathology
Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....

.

Innate

The innate
Innate immune system
The innate immune system, also known as non-specific immune system and secondary line of defence, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner...

 immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...

s 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.

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
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

 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. 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.

Cell-mediated

The cell-mediated
Cell-mediated immunity
Cell-mediated immunity is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural killer cells , antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen...

 response to the virus and to vectors is poorly characterised and has been largely ignored in the literature as recently as 2005. Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring. Combined with data that shows that CD8+ T-cells
Cytotoxic T cell
A cytotoxic T cell belongs to a sub-group of T lymphocytes that are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses , or are otherwise damaged or...

 can recognise elements of the AAV capsid in vitro, 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
T helper cell
T helper cells are a sub-group of lymphocytes, a type of white blood cell, that play an important role in the immune system, particularly in the adaptive immune system. These cells have no cytotoxic or phagocytic activity; they cannot kill infected host cells or pathogens. Rather, they help other...

 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. A number of candidate T cell stimulating epitope
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...

s 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.

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
    Cell membrane
    The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...

  2. endocytosis
    Endocytosis
    Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...

  3. endosomal trafficking
  4. escape from the late endosome
    Endosome
    In biology, an endosome is a membrane-bound compartment inside eukaryotic cells. It is a compartment of the endocytic membrane transport pathway from the plasma membrane to the lysosome. Molecules internalized from the plasma membrane can follow this pathway all the way to lysosomes for...

     or lysosome
    Lysosome
    thumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...

  5. translocation to the nucleus
    Cell nucleus
    In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...

  6. formation of double-stranded DNA replicative form of the AAV genome
  7. rep genes expression
  8. genome replication
    DNA replication
    DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...

  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
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...

 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
Hydroxyurea
Hydroxycarbamide or hydroxyurea is an antineoplastic drug, first synthesized in 1869, used in myeloproliferative disorders, specifically polycythemia vera and essential thrombocythemia...

.

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).

External links

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