Adeno-associated viruses (AAV) are small

virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsk ...

es that infect humans and some other primate species. They belong to the

genus Genus ( plural genera ) is a taxonomic rank used in the biological classification of living and fossil organisms as well as viruses. In the hierarchy of biological classification, genus comes above species and below family. In binomial nom ...

Dependoparvovirus ''Dependoparvovirus'' (formerly ''Dependovirus'' or Adeno-associated virus group) is a genus in the subfamily '' Parvovirinae'' of the virus family '' Parvoviridae''; they are Group II viruses according to the Baltimore classification. Some dep ...

'', which in turn belongs to the

family Family (from la, familia) is a group of people related either by consanguinity (by recognized birth) or affinity (by marriage or other relationship). The purpose of the family is to maintain the well-being of its members and of society. Idea ...

Parvoviridae Parvoviruses are a family of animal viruses that constitute the family ''Parvoviridae''. They have linear, single-stranded DNA (ssDNA) genomes that typically contain two genes encoding for a replication initiator protein, called NS1, and the pr ...

''. They are small (approximately 26 nm in diameter) replication-defective, nonenveloped viruses and have linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). AAV are not currently known to cause

disease A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...

. The viruses cause a very mild

immune response An immune response is a reaction which occurs within an organism for the purpose of defending against foreign invaders. These invaders include a wide variety of different microorganisms including viruses, bacteria, parasites, and fungi which could ...

. Several additional features make AAV an attractive candidate for creating viral vectors for

gene therapy Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...

, and for the creation of

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 s ...

Gene therapy Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...

vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding g ...

of the host cell. In the native virus, however, integration of virally carried genes into the host genome does occur. Integration can be important for certain applications, but can also have unwanted consequences. Recent human clinical trials using AAV for gene therapy in the retina have shown promise.

History

The adeno-associated virus (AAV), previously thought to be a contaminant in adenovirus preparations, was first identified as a

dependoparvovirus ''Dependoparvovirus'' (formerly ''Dependovirus'' or Adeno-associated virus group) is a genus in the subfamily '' Parvovirinae'' of the virus family '' Parvoviridae''; they are Group II viruses according to the Baltimore classification. Some dep ...

in the 1960s in the laboratories of Bob Atchison at Pittsburgh and Wallace Rowe at

NIH The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the late ...

. Serological studies in humans subsequently indicated that, despite being present in people infected by helper viruses such as adenovirus or herpes virus, AAV itself did not cause any disease.

Use in gene therapy

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 Chromosome 19 is one of the 23 pairs of chromosomes in humans. People normally have two copies of this chromosome. Chromosome 19 spans more than 58.6 million base pairs, the building material of DNA. It is considered the most gene-rich chromosom ...

. This 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 Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...

. 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 (ITRs) that aid in

concatemer A concatemer is a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series. These polymeric molecules are usually copies of an entire genome linked end to end and separated by ''cos'' sites (a protein bi ...

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 Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted: * Wanted immunogenicity typically relates to vaccines, where the injectio ...

, seemingly restricted to generation of neutralizing

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

, while they induce no clearly defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for human

. 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 concatemers, 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. Recent reports suggest that scAAV vectors are more immunogenic than single stranded adenovirus vectors, inducing a stronger activation of

cytotoxic T lymphocytes A cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected by intracellular pa ...

. Humoral immunity instigated by infection with the wild type is thought to be common. The associated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications. Accordingly, the majority of clinical trials under way involve delivery of AAV2 into the brain, a relatively immunologically privileged organ. In the brain, AAV2 is strongly neuron-specific.

Clinical trials

As of 2019, AAV vectors have been used in over 250 clinical trials worldwide, approximately 8.3% of virus-vectored gene-therapy trials. Recently, promising results have been obtained from Phase 1 and Phase 2 trials for a number of diseases, including

Leber's congenital amaurosis Leber congenital amaurosis (LCA) is a rare inherited eye disease that appears at birth or in the first few months of life. It affects about 1 in 40,000 newborns. LCA was first described by Theodor Leber in the 19th century. It should not be co ...

, hemophilia,

congestive heart failure Heart failure (HF), also known as congestive heart failure (CHF), is a syndrome, a group of signs and symptoms caused by an impairment of the heart's blood pumping function. Symptoms typically include shortness of breath, excessive fatigue, ...

, spinal muscular atrophy, lipoprotein lipase deficiency, and

Parkinson's disease Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

Fundamental Biology

Genomics, transcriptomics and proteomics

The AAV genome is built of single-stranded deoxyribonucleic acid (ss DNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises ITRs at both ends of the DNA strand, and two

open reading frame In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible readin ...

s (ORFs): ''rep'' and ''cap''. The former is composed of four

overlapping genes An overlapping gene (or OLG) is a gene whose expressible nucleotide sequence partially overlaps with the expressible nucleotide sequence of another gene. In this way, a nucleotide sequence may make a contribution to the function of one or more gen ...

encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of

capsid A capsid is the protein shell of a virus, enclosing its genetic material. It consists of several oligomeric (repeating) structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may ...

proteins: VP1, VP2 and VP3, which interact to form a capsid with 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. The feature of these sequences that gives them this property is their ability to form a

hairpin A hairpin or hair pin is a long device used to hold a person's hair in place. It may be used simply to secure long hair out of the way for convenience or as part of an elaborate hairstyle or coiffure. The earliest evidence for dressing the hai ...

, which contributes to so-called self-priming that allows

primase DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some living organisms) segment called a primer complementary to a ssDNA (single-stranded ...

-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 {{Short pages monitor # attachment to the

cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...

# receptor-mediated endocytosis # endosomal trafficking # escape from the late

endosome Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can ...

lysosome A lysosome () is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane pr ...

# translocation to the

nucleus Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to: *Atomic nucleus, the very dense central region of an atom * Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA Nucl ...

# uncoating # formation of double-stranded DNA replicative form of the AAV genome # expression of ''rep'' genes # genome replication # expression of ''cap'' genes, synthesis of progeny ssDNA particles # assembly of complete

virion A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's ...

s, and # 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 The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...

phase. The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. Adeno-associated virus spreads by co-infecting a cell with a helper virus. The first helper virus 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 vaccinia, or by genotoxic agents, such as UV irradiation or

hydroxyurea Hydroxycarbamide, also known as hydroxyurea, is a medication used in sickle-cell disease, essential thrombocythemia, chronic myelogenous leukemia, polycythemia vera, and cervical cancer. In sickle-cell disease it increases fetal hemoglobin and d ...

. Depending on the presence or absence of a helper virus, the life cycle of AAV follows either a lytic or lysogenic pathway, respectively. If there is a helper virus, AAV's gene expression activates, allowing the virus to replicate using the host cell's polymerase. When the helper virus kills the host cell, the new AAV virions are released. If there is not a helper virus present, AAV exhibits lysogenic behavior. When AAV infects a cell alone, its gene expression is repressed (AAV does not replicate), and its genome is incorporated into the host genome (into human chromosome 19). In rare cases, lysis can occur without a helper virus, but usually AAV can not replicate and kill a cell on its own. 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).

References

External links

* * {{DEFAULTSORT:Adeno-Associated Virus Applied genetics Experimental cancer treatments Parvovirinae Parvoviruses