In , a human bocavirus was characterized in respiratory secretions of Swedish children 2 and has been associated with lower respiratory tract infections worldwide, often in combination with other viral infections 1 , 21 , 35 , 38 , Related parvoviruses named human bocaviruses 2 to 4 have also been detected in the feces of children with acute flaccid paralysis 39 , 45 and diarrhea 7.
Members of the Bocavirus genus also infect bats GenBank accession no. JQ , cows 17 , dogs 42 , 63 , pigs 11 , 19 , 24 , 48 , 64 , chimpanzees 67 , and gorillas Human parvovirus 4 PARV4 was initially described in in the plasma of a febrile injection drug user Because of the high prevalence of anti-PARV4 antibodies in hemophiliacs using plasma pool-derived coagulation factors and in hepatitis C virus HCV - and HIV-positive injection drug users, the transmission of PARV4 in developed countries is thought to be mainly parenteral 29 , 54 , 66 , 72 , Related viruses have been detected in chimpanzees 67 , baboons 67 , bats 16 , sheep 69 , pigs 47 , and cows 47 , and a Parvoviridae genus named Partetravirus has been proposed PARV4 has been reported in patients with different symptoms, but the full extent of its pathogenicity remains uncertain 8 , 18 , 36 , Here we report on a viral metagenomic analysis of feces of children from Burkina Faso with acute diarrhea 12 allowing the characterization of their enteric viruses, including the genetic characterization of novel parvoviruses from a previously unrecognized genus 12 , Samples were collected between November and February from the capital city of Ouagadougou in Burkina Faso Rotavirus antigen-negative samples were used in this study approved by the University of California at San Francisco committee on human research.
Viral particles were first enriched by filtration and nuclease treatment to digest non-particle-protected nucleic acids 3. Each of 49 molecular tags was used on two fecal samples, for a total of 98 diarrhea samples. Because fecal samples were analyzed in pairs, the viruses identified are reported per sample pair rather than for individual samples. Primers BF. Viral particles in fecal samples from children with acute diarrhea were first enriched by filtration. Non-particle-protected nucleic acids were digested with nucleases to reduce the nonviral background, and the remaining nucleic acids were extracted, amplified by random RT-PCR, tagged, and then pyrosequenced, generating over half a million reads see Materials and Methods.
The distributions of these viral hits among the 49 pairs of samples analyzed are shown in Table 1. Because diarrhea samples were pooled in pairs prior to random RT-PCR, viruses detected could only be assigned to pairs of samples. From 0 to 6 eukaryotic viruses average, 1. Distribution of sequence reads to different viral species and total of human viruses in 49 sample pairs.
A sequence identity matrix was then generated Table 2. Bufavirus genome and phylogeny. A Organization of the bufavirus genome. The PLA 2 similarity region, including the calcium-binding region and catalytic residues, is shown. Theoretical splicing for expression of VP1 is shown. B Pairwise sliding window of percent nucleotide identity of bufavirus aligned with representatives of other parvovirus genera and bufavirus 2.
Genetic distances were calculated by Kimura's two-parameter method PHYLIP , and a phylogenetic tree with bootstrap resampling of the alignment data sets was generated using the neighbor-joining method. Each scale indicates the number of amino acid substitutions per position.
Proposed and official Parvoviridae genera are labeled. The names and accession numbers of the taxa used are listed in Table S1 in the supplemental material. The phospholipase catalytic residues HD and D were present at amino acid positions 40 to 41 and 62 Fig. The VP2 protein was most closely related to those of amdoviruses Table 2.
Such repeated sequences were not reported in other parvoviruses. The three major proteins of bufavirus were aligned with those of other parvoviruses and phylogenetically analyzed Fig. Bufavirus clustered with the members of the Parvovirus genus in NS1 but was basal to both the Parvovirus and Amdovirus genera in its capsid region.
To investigate the prevalence of this novel parvovirus, nested PCR primers targeting the NS1 region were designed. PCR amplicons were directly sequenced to confirm their identification. The complete protein-coding regions of these three related viral genomes were then determined. The coding regions of two strains, BF7 GenBank accession no. We therefore provisionally named this genome bufavirus 2-BF39 to reflect the presence of a second tentative species highly divergent in its VP2 protein sequence.
The high degree of identity between the two species in their NS genes relative to their more divergent capsid genes may indicate that the VP region was acquired by recombination from a still uncharacterized viral genome or that the structural region diverged at a much higher rate than the nonstructural region.
An alignment of NS1 and VP1 from both bufavirus species with the most closely related parvoviruses is shown in Fig. S1 in the supplemental material. One bufavirus 1 was amplified from the feces of one Tunisian child, indicating that these viruses are not geographically restricted. The detection of two samples containing rotavirus sequences despite the exclusion of rotavirus-positive samples likely reflects viral loads below the assay's antigen detection level.
The metagenomic detection of bufavirus sequences in only one sample while three more samples were positive by nested PCR reflects the lower sensitivity of pyrosequencing at the depth used here. The rate of infection with other viruses measured here by metagenomics is therefore likely also an underestimate of their actual prevalence in feces. The genetic characterization of a second Bufavirus species with a highly divergent capsid gene indicates that wider geographic sampling for related viruses will likely reveal other related species.
The genetic diversity within this proposed genus may also indicate a range of phenotypes upon their hosts. Wider geographic sampling of human and animal fecal samples will provide a better description of the genetic diversity of this proposed Parvoviridae genus.
Serological assays will help determine whether bufaviruses infect humans or are simply passing through the gut from a dietary source. Case-control studies comparing the presence and viral loads of bufavirus DNA in feces or the prevalence of anti-bufavirus IgM will also help determine whether members of this viral clade are associated with diarrhea or other symptoms. Read article at publisher's site DOI : Microbiome , 9 1 , 15 Dec Virol J , 18 1 , 24 Oct Pathogens , 10 9 , 07 Sep Vaccines Basel , 9 7 , 23 Jun Viruses , 13 6 , 04 Jun This data has been text mined from the article, or deposited into data resources.
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Europe PMC requires Javascript to function effectively. Try out PMC Labs and tell us what you think. Learn More. There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus AAV is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies.
The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.
The discovery of DNA as the biomolecule of genetic inheritance and disease opened up the prospect of therapies in which mutant, damaged genes could be altered for the improvement of the human condition. The recent ability to rapidly and affordably perform human genetics on hundreds of thousands of people, and to sequence complete genomes, has resulted in an explosion of nucleic acid sequence information and has allowed us to identify the gene, or genes, that might be driving a particular disease state.
This concept seems particularly true for the treatment of monogenic diseases, i. This seemingly simple premise has been the goal of gene therapy for over 40 years.
Until relatively recently, that simple goal was very elusive as technologies to safely deliver nucleic acid cargo inside cells have lagged behind those used to identify disease-associated genes. One of the earliest approaches investigated was the use of viruses, naturally occurring biological agents that have evolved to do one thing, i. There are numerous viral agents that could be selected for this purpose, each with some unique attributes that would make them more or less suitable for the task, depending on the desired profile [ 1 ].
However, the undesired properties of some viral vectors, including their immunogenic profiles or their propensity to cause cancer have resulted in serious clinical adverse events and, until recently, limited their current use in the clinic to certain applications, for example, vaccines and oncolytic strategies [ 2 ].
More artificial delivery technologies, such as nanoparticles, i. Not surprisingly, they also have encountered some unwanted safety signals that need to be better understood and controlled [ 3 ]. Adeno-associated virus AAV is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations [ 4 , 5 ], hence its name.
AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing.
These coding sequences are flanked by inverted terminal repeats ITRs that are required for genome replication and packaging. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of VP1:VP2:VP3 [ 6 ].
The aap gene encodes the assembly-activating protein AAP in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly [ 7 ]. Although there is much more to the biology of wild-type AAV, much of which is not fully understood, this is not the form that is used to generate gene therapeutics.
In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [ 9 ]. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell.
These characteristics make rAAV ideal for certain gene therapy applications. Following is an overview of the practical considerations for the use of rAAV as a gene therapy agent, based on our current understanding of viral biology and the state of the platform. The final section provides an overview for how rAAV has been incorporated into clinical-stage gene therapy candidates, as well as the lessons learned from those studies that can be applied to future therapeutic opportunities.
The main point of consideration in the rational design of an rAAV vector is the packaging size of the expression cassette that will be placed between the two ITRs. As a starting point, it is generally accepted that anything under 5 kb including the viral ITRs is sufficient [ 10 ]. Attempts at generating rAAV vectors exceeding packaging cassettes in excess of 5 kb results in a considerable reduction in viral production yields or transgene recombination truncations [ 11 ].
As a result, large coding sequences, such as full-length dystrophin, will not be effectively packaged in AAV vectors. Therefore, the use of dual, overlapping vector strategies reviewed by Chamberlain et al.
An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes. After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene, which is considered a limiting step in the onset of transgene expression [ 13 ].
An alternative is to use self-complementary AAV, in which the single-stranded packaged genome complements itself to form a double-stranded genome in the nucleus, thereby bypassing that process [ 13 , 14 ]. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 3.
AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator Fig. In many cases, strong, constitutively active promoters are desired for high-level expression of the gene of interest.
All of these promoters provide constitutively active, high-level gene expression in most cell types. Some of these promoters are subject to silencing in certain cell types, therefore this consideration needs to be evaluated for each application [ 16 ].
Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. Although many therapeutic strategies involve systemic delivery, it is often desirable to have cell- or tissue-specific expression. Likewise, for local delivery strategies, undesired systemic leakage of the AAV particle can result in transduction and expression of the gene of interest in unwanted cells or tissues.
Likewise, the neuron-specific enolase promoter can attain high levels of neuron-specific expression [ 18 , 19 ]. Often is the case, systemic delivery of AAV results in a significant accumulation in the liver. While this may be desirable for some applications, AAV can also efficiently transduce other cells and tissues types. Finally, there are now technologies that have the ability to generate novel, tissue-specific promoters, based on DNA regulatory element libraries [ 22 ].
Over the course of the past 10—15 years, much work has been done to understand the correlation between codon usage and protein expression levels.
Although bacterial expression systems seem to be most affected by codon choice, there are now many examples of the effects of codon engineering on mammalian expression [ 23 ].
Many groups have developed their own codon optimization strategies, and there are many free services that can similarly provide support for codon choice.
Codon usage has also been shown to contribute to tissue-specific expression, and play a role in the innate immune response to foreign DNA [ 24 , 25 ]. With regard to the gene of interest, codon engineering to support maximal, tissue-specific expression should be performed. Although there is much known about these individual components that needs to be considered when designing an AAV vector, the final design will most likely need to be determined empirically.
It is not yet possible to know how a particular design will function by just combining the best elements together based on published reports, therefore considerable trial and error will eventually be required for deciding on the final construct. In addition, one also needs to consider the differences between in vitro and in vivo activity. Although it is possible to model rAAV expression in rodents, there is still significant concern about the translatability to humans.
AAV has evolved to enter cells through initial interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose and heparin sulfate [ 29 , 30 ]. Subtle differences in sugar-binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [ 31 — 33 ].
For example, AAV9 has a preference for primary cell binding through galactose as a result of unique amino acid differences in its capsid sequence [ 34 ]. It has been postulated that this preferential galactose binding could confer AAV9 with the unique ability to cross the blood—brain barrier BBB and infect cells of the CNS, including primary neurons [ 35 , 36 ].
In addition to the primary carbohydrate interactions, secondary receptors have been identified that also play a role in viral transduction and contribute to cell and tissue selectivity of viral variants. As a result of these subtle variations in primary and secondary receptor interactions for the various AAV variants, one can choose a variant that possesses a particular tropism and preferentially infects one cell or tissue type over others Table 1.
For example, AAV8 has been shown to effectively transduce and deliver genes to the liver of rodents and non-human primates, and is currently being explored in clinical trials to deliver genes for hemoglobinopathies and other diseases [ 38 ]. Likewise, AAV1 and AAV9 have been shown to be very effective at delivering genes to skeletal and cardiac muscle in various animal models [ 39 — 46 ]. Engineered AAV1 is currently being explored as the gene transfer factor in clinical trials for heart failure, and has been approved for the treatment of lipoprotein lipase deficiency [ 47 ].
However, although different AAV vectors have been identified that preferentially transduce many different cell types, there are still cell types for which AAV has proven difficult to transduce. With the strong desire to utilize AAV to deliver genes to very selective cell and tissue types, efforts to clone novel AAV variants from human and primate tissues have identified a number of unique capsid sequences that are now being studied for tropism specificities [ 48 ].
In addition, recombinant techniques involving capsid shuffling, directed evolution, and random peptide library insertions are being utilized to derive variants of known AAVs with unique attributes [ 49 — 51 ]. In vivo-directed evolution has been successfully used to identify novel AAV variants that preferentially transduce the retinal cells of the eye, as well as other cell populations, including those in the CNS [ 50 , 52 , 53 ]. In addition, these techniques have been employed to identify novel AAV variants with reduced sensitivities to neutralizing antibodies NAbs [ 54 — 57 ].
Alternatively, other investigators have inserted larger binding proteins into different regions of AAV capsid proteins to confer selectivity. For example, DARPins designed ankyrin repeat proteins , portions of protein A, and cytokines, have all been engineered into the capsid of AAV for the purpose of greater cell specificity and targeting [ 58 , 59 ].
As we continue to learn more about the biology of AAV with regard to the mechanisms involved in membrane translocation, endosomal escape, and nuclear entry, we will undoubtedly find opportunities to engineer unique properties into viral vectors through modulating one or more of these functions.
For example, it has been hypothesized that surface-exposed serine and tyrosine residues could be phosphorylated upon viral cell entry, resulting in their ubiquitination and proteolytic degradation [ 62 — 64 ].
Studies have shown that mutation of tyrosine to phenylalanine, which prevents this phosphorylation, results in dramatically improved transduction efficiencies [ 63 ]. Similar efforts have been made in attempts to limit the effects of NAbs, as discussed below.
As one gives careful consideration to these selection criteria, it is possible to narrow the choices of which AAVs natural or engineered to profile. Alternatively, one can begin the path of exploring fully engineered versions of AAV for truly selective cell targeting and optimized transduction.
Because our understanding of AAV biology is in relative infancy, many of these efforts will remain empirical for quite some time as optimization for one activity could have a negative impact on another.
Nonetheless, the future looks promising for this highly adaptable platform. One of the appealing aspects of using rAAV as a gene transfer vector is that it is composed of biomolecules, i. Fortunately, a full-package virus lacks engineered lipids or other chemical components that could contribute to unwanted toxicities or immunogenicities that may not be predictable or fully understood.
In general, AAV has been shown to be less immunogenic than other viruses. Although not completely understood, one possible reason for this may hinge on the observation that certain AAVs do not efficiently transduce antigen-presenting cells APCs [ 65 ]. Additionally, unlike previous viral delivery strategies, rAAV does not contain any viral genes, therefore there will be no active viral gene expression to amplify the immune response [ 66 ].
Although AAV has been shown to be poorly immunogenic compared with other viruses i. This is further complicated by the fact that most people have already been exposed to AAV and have already developed an immune response against the particular variants to which they had previously been exposed, resulting in a pre-existing adaptive response.
It should be of no surprise that the formidable challenge is how to deliver a therapeutically efficacious dose of rAAV to a patient population that already contains a significant amount of circulating NAbs and immunological memory against the virus [ 67 ]. Whether administered locally or systemically, the virus will be seen as a foreign protein, hence the adaptive immune system will attempt to eliminate it.
This leads to plasma cell and memory cell development that has the capacity to secrete antibodies to the AAV capsid. These antibodies can either be neutralizing, which has the potential to prevent subsequent AAV infection, or non-neutralizing.
Non-NAbs are thought to opsonize the viral particles and facilitate their removal through the spleen [ 70 ]. Upon entry of the virus into target cells during the course of the natural infection process, the virus is internalized through clathrin-mediated uptake into endosomes [ 71 ]. After escape from the endosome, the virus is transported to the nucleus where the ITR-flanked transgene is uncoated from the capsid [ 72 ].
The pathway and mechanism of AAV intracellular transport and processing is not fully understood, and there are quite a few areas of debate with regard to current understanding. The most current hypothesis is that following endosomal escape, capsid breakdown and uncoating occurs after subsequent nuclear translocation. However, it is thought that cytosolic ubiquitination of the intact virus can occur during transport to the nucleus [ 73 ]. This would be a critical step in directing capsid proteins to the proteasome for proteolytic processing into peptides for class I MHC presentation.
This hypothesis is supported by data in which proteasome inhibitors, or mutations in capsid residues that are sites for ubiquitination, can limit class I presentation and T-cell activation [ 73 — 76 ]. However, apparent differences have been observed for T-cell activation to different AAV variants with significant sequence identity. At this time, it is unclear whether this is due to subtle capsid sequence differences and susceptibility to MHC I presentation or differential cellular processing that is innate to the different AAV variants, or simply due to contaminants in vector preparations [ 76 ].
In addition to an adaptive immunological reaction to the capsid of AAV, the transgene can elicit both an adaptive and an innate response. If the transgene encodes a protein that can be recognized as foreign, it too can generate a similar B- and T-cell response. For example, in replacement therapy applications in which the protein to be replaced is the consequence of a null genotype, the immune system will have never selected against precursor B and T cells to that protein [ 70 , 77 ].
Likewise, if the transgene is an engineered variant, the engineered sequence can be recognized as foreign. Even the variable regions of antibodies can activate an adaptive response that can result in deletion of target cells that are expressing transgene as a result of AAV delivery. Finally, a transgene with a significant number of CpG dinucleotides can activate innate responses through toll-like receptor TLR molecular pattern receptors [ 78 ].
To date, this represents one of the biggest therapeutic challenges to the use of systemically delivered AAV, and is thought to be one of the factors in early clinical failures [ 79 ].
Pre-existing immunity to AAV can often be overcome by selecting a particular AAV variant that has not circulated throughout the human population, and, therefore, does not have any memory responses elicited against it, including NAbs and T cells [ 80 ]. Additionally, some of the AAV evolution technologies discussed above have been used to identify AAVs that are resistant to the effects of NAbs [ 50 , 57 ].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
National Center for Biotechnology Information , U. PLoS One. Published online Sep 4. Olive T. Leo L. Dhanasekaran Vijaykrishna, Editor. Author information Article notes Copyright and License information Disclaimer. Competing Interests: The authors have declared that no competing interests exist.
Received May 9; Accepted Jul This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
This article has been cited by other articles in PMC. Figure S2: Alignment of N-ternimal amino acid upper and nucleotide lower sequences containing start and stop codons of two pigeon Mesiviruses. Figure S3: Alignment of VP6 proteins of the novel pigeon rotavirus and other closely-related rotavirus species. Figure S4: Phylogenetic trees of structural shaded box and non-structural proteins of the novel pigeon rotavirus and other rotavirus species A-H.
Table S3: Representative members in the subfamily Parvovirinae for the phylogenetic trees in Figure 1 and their GenBank numbers. Table S4: Representative members in the family Picornaviridae for the phylogenetic tree in Figure 2 and their GenBank numbers. Abstract Birds are frequent sources of emerging human infectious diseases. Introduction Many infectious diseases in humans are caused by pathogens originating from a wide variety of animals. Bioinformatics Analysis We received Open in a separate window.
Figure 1. Pigeon parvovirus genome and phylogeny. Pigeon Picornavirus Picornaviruses are small, non-enveloped, single-stranded RNA viruses whose prototype is poliovirus. Figure 2. Mesivirus genome and phylogeny. Pigeon Rotavirus Rotaviruses consist of at least eight groups or species A through H with multiple P and G genotypes which together comprise a genus in the family Reoviridae. Figure 3. Pigeon rotavirus VP6 and phylogeny. Figure 4. Pigeon rotavirus VP4, VP7 and phylogeny.
GenBank numbers of these viruses are available in Table S5. Discussion Metagenomics has been used to analyze viral nucleic acids in feces collected from humans and a growing range of animals including primates, horse, bats, rodents, pigs, dogs, and turkeys [42] , [58] , [65] , [69] , [89] — [92]. PDF Click here for additional data file. Figure S2 Alignment of N-ternimal amino acid upper and nucleotide lower sequences containing start and stop codons of two pigeon Mesiviruses.
Figure S3 Alignment of VP6 proteins of the novel pigeon rotavirus and other closely-related rotavirus species. Figure S4 Phylogenetic trees of structural shaded box and non-structural proteins of the novel pigeon rotavirus and other rotavirus species A-H.
Table S3 Representative members in the subfamily Parvovirinae for the phylogenetic trees in Figure 1 and their GenBank numbers. Table S4 Representative members in the family Picornaviridae for the phylogenetic tree in Figure 2 and their GenBank numbers. References 1. Curr Opin Virol. Rev Sci Tech 23 : — Emerg Infect Dis 16 : 1—7. Emerg Infect Dis 13 : 6— Rev Sci Tech 19 : — J Prev Med Hyg 52 : — Rumschlag-Booms E, Rong L Influenza a virus entry: implications in virulence and future therapeutics.
Adv Virol : Avian Dis 54 : — Ann Acad Med Singapore 37 : — Lubroth J Control strategies for highly pathogenic avian influenza: a global perspective. Dev Biol Basel : 13— Reisen WK Epidemiology of St. Louis encephalitis virus. Adv Virus Res 61 : — Ann N Y Acad Sci : — Acta Virol 53 : — Avian Pathol 33 : — J Vet Med Sci 74 : — Avian Dis 55 : — Nat Rev Microbiol 11 : — Epidemiol Infect : — Annu Rev Entomol 53 : 61— West nile virus disease and other arboviral diseases - United States, Integr Zool 6 : — Emerg Infect Dis 10 : — In the only clearly documented instance of therapeutic correction of an inborn error, the inherent oncogenic properties of the original virus Moloney murine leukemia virus were retained; 4 of 12 patients with X-linked severe combined immunodeficiency disease developed leukemia.
In this experiment, bone marrow precursor cells were transduced and allowed to differentiate. Under these conditions, a vector that would integrate was needed.
Among current viral vectors, only those derived from retroviruses have the ability to integrate at a reasonable frequency; retroviruses require cell division for integration to occur, whereas lentiviruses and foamy viruses can enter the nucleus and integrate in nondividing cells. Lentiviral vectors carry the psychological burden of being derived from significant pathogens, but foamy viruses infect a high percentage of humans without having been implicated as the cause of disease.
Although there are production challenges, very promising results have been obtained in a canine model of congenital granulomatosis. The overriding theoretical consideration is that retroviruses integrate at many sites in the human genome, so there is always the concern of insertional mutagenesis possibly causing oncogenesis. AAV-2, and presumably other serotypes, has been reported to integrate at a specific site in the q arm of chromosome 19 AAVS1.
This exceeds the frequency that has been observed, with the most successful vectors being derived from bacteriophage systems. As discussed above Future Prospects , it is possible to design AAV vectors that can integrate in a site-specific manner; therefore, a DNA virus vector is possible. AAV was initially considered as a vector by only a few laboratories.
This undoubtedly reflected the lack of familiarity with the virus, since it is nonpathogenic and, thus, of interest only to those inherently interested in its distinctive biology. However, as noted above, with time, it has become among the most commonly used viral vectors.
This is likely the consequence of several factors. First, almost all other viral vectors lead to an initial burst of transgene expression that commonly disappears after a relatively short time, measured in weeks. AAV transgene expression, on the other hand, frequently persists for years or the life time of the animal model. Second, other viral vectors have a greater capacity with which to insert the transgene s.
However, with time and clever engineering, it has been possible to insert originally very large transgenes into AAV vectors. Interestingly, it has also proved to be feasible to have split vectors in which one construct has slight sequence overlap with a second construct so that recombination after vector nuclear entry leads to the intact transgene product being expressed.
Thus, the consequences of the small size of the AAV genome have been overcome to a large extent. Another significant positive feature of AAV vectors is that they frequently do not elicit a deleterious immune response. This feature is dependent on the site of administration and the effective MOI of the vector used.
Another factor is that AAV appears to be taken up poorly by dendritic cells. Finally, the small capacity of the genome has meant that no viral genes remain. In a parallel manner, the latest version of adenovirus vectors is the gutless vectors from which all viral genes have also been removed. Interestingly, the gutless adenovirus vectors still do not perform as well as AAV vectors in terms of expression persistence.
It is tempting to speculate that the difference reflects the special structure of the AAV ITR, which could serve both as an insulator and to protect against cellular exonucleases. Thus, AAV has become appreciated as a good vector for the transduction of postmitotic cells. At this time, retroviral vectors remain the vector of choice for the transduction of stem or progenitor cells despite the inherent concern of possible oncogenesis. These considerations apply for situations in which long-term transgene expression is desired.
In cases such as immunization or vector-induced oncolysis, where expression at higher levels for relatively short periods of time is desirable, other viral vectors such as those derived from adenovirus and herpesvirus have more useful characteristics. What has become apparent is that different vectors have characteristics that are advantageous in specific cases. Thus, the notion of best vector depends on the question of what is best for what purpose.
AAV remains a promising delivery system for the realization of the dream of gene therapy. It compares favorably to other viral vectors, especially when sustained transgene expression is desired.
Although nonviral vector systems such as lipid-mediated vectors, hydrodynamic delivery, and the gene gun have been advocated and tried, to date, none have approached the efficacy of the viral delivery systems. Whether such development will occur remains unknown. AAV vectors have achieved some success, and it seems likely that some of the advances described above and others not yet envisioned will enable AAV to become an effective therapeutic agent.
National Center for Biotechnology Information , U. Journal List Clin Microbiol Rev v. Clin Microbiol Rev. Shyam Daya 1 and Kenneth I. Kenneth I. Author information Copyright and License information Disclaimer. Mailing address: P. Phone: Fax: E-mail: ude. This article has been cited by other articles in PMC. Abstract Summary: The unique life cycle of adeno-associated virus AAV and its ability to infect both nondividing and dividing cells with persistent expression have made it an attractive vector.
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