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Adeno-associated virus: from defective virus to effective vector.

Gonçalves MA - Virol. J. (2005)

Bottom Line: The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology.In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed.In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands. m.goncalves@lumc.nl

ABSTRACT
The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

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Diagram of the generation and transduction of a self-complementary AAV vector as compared to that of a conventional recombinant AAV. Left panel: According to the AAV DNA replication scheme, full-length rAAV genomes of both polarities are generated from duplex monomeric (DM) and duplex dimeric (DD) replicative intermediates and individually packaged in AAV capsids. In the nucleus of transduced cells the single-stranded genomes can either be a target for degradation or be converted into transcriptionally active double-stranded templates. The single-to-double strand DNA conversion depends on complementary chain synthesis or on the recruitment of a complementary genome (i.e., intermolecular hybridization). Right panel: According to the same replication model, a rAAV genome with roughly half the size of the wild-type AAV DNA and with one trs mutated, generates DD replicative intermediates with an inverted repeat configuration containing wild-type ITRs at the extremities and mutated ITRs at the axis of symmetry. Single-stranded molecules derived from these DNA structures are packaged in AAV capsids. After uncoating in the target cell nucleus, these molecules can readily fold into double-stranded templates through intramolecular base pairing due to their self-complementary nature (i.e., intramolecular hybridization).
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Figure 6: Diagram of the generation and transduction of a self-complementary AAV vector as compared to that of a conventional recombinant AAV. Left panel: According to the AAV DNA replication scheme, full-length rAAV genomes of both polarities are generated from duplex monomeric (DM) and duplex dimeric (DD) replicative intermediates and individually packaged in AAV capsids. In the nucleus of transduced cells the single-stranded genomes can either be a target for degradation or be converted into transcriptionally active double-stranded templates. The single-to-double strand DNA conversion depends on complementary chain synthesis or on the recruitment of a complementary genome (i.e., intermolecular hybridization). Right panel: According to the same replication model, a rAAV genome with roughly half the size of the wild-type AAV DNA and with one trs mutated, generates DD replicative intermediates with an inverted repeat configuration containing wild-type ITRs at the extremities and mutated ITRs at the axis of symmetry. Single-stranded molecules derived from these DNA structures are packaged in AAV capsids. After uncoating in the target cell nucleus, these molecules can readily fold into double-stranded templates through intramolecular base pairing due to their self-complementary nature (i.e., intramolecular hybridization).

Mentions: Another development in rAAV design is the so-called self-complementary AAV vectors (scAAV) [94]. The scAAV approach builds on the ability of AAV to package replicons with half the size of the wild-type DNA in the form of single-stranded dimeric genomes with an inverted repeat configuration [95]. In the target cell, these self-complementary molecules can readily fold back into double-stranded forms without the need for de novo DNA synthesis or for the annealing of sense and antisense strands (Fig. 6). Ultimately, regardless of the mechanism(s) at play, scAAV lead to enhanced formation of transcription-competent double-stranded genomes thus improving the expression kinetics and yields of vector-encoded products. This scAAV method was subsequently perfected by mutagenesis of one of the two trs sequences to force the generation of dimeric over monomeric replicative forms (Fig. 6) [96]. The main disadvantage of this approach is the need to limit the size of the transgenes that can be delivered to approximately half the length of the already small AAV genome. It is conceivable that this drawback can be tackled by coupling scAAV with heterodimerization strategies. Alternatively, long double-stranded rAAV genomes can be transferred into target cells via capsids of larger viruses such as Ad [97-100], baculovirus [101] or HSV [102]. In some of these hybrid viral vector systems, integration of the rAAV DNA into the AAVS1 locus on human chromosome 19 was accomplished by transient expression of AAV Rep activities in the target cells [38]. Targeted DNA integration is advantageous since it dispels the insertional oncogenesis concerns discussed above.


Adeno-associated virus: from defective virus to effective vector.

Gonçalves MA - Virol. J. (2005)

Diagram of the generation and transduction of a self-complementary AAV vector as compared to that of a conventional recombinant AAV. Left panel: According to the AAV DNA replication scheme, full-length rAAV genomes of both polarities are generated from duplex monomeric (DM) and duplex dimeric (DD) replicative intermediates and individually packaged in AAV capsids. In the nucleus of transduced cells the single-stranded genomes can either be a target for degradation or be converted into transcriptionally active double-stranded templates. The single-to-double strand DNA conversion depends on complementary chain synthesis or on the recruitment of a complementary genome (i.e., intermolecular hybridization). Right panel: According to the same replication model, a rAAV genome with roughly half the size of the wild-type AAV DNA and with one trs mutated, generates DD replicative intermediates with an inverted repeat configuration containing wild-type ITRs at the extremities and mutated ITRs at the axis of symmetry. Single-stranded molecules derived from these DNA structures are packaged in AAV capsids. After uncoating in the target cell nucleus, these molecules can readily fold into double-stranded templates through intramolecular base pairing due to their self-complementary nature (i.e., intramolecular hybridization).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1131931&req=5

Figure 6: Diagram of the generation and transduction of a self-complementary AAV vector as compared to that of a conventional recombinant AAV. Left panel: According to the AAV DNA replication scheme, full-length rAAV genomes of both polarities are generated from duplex monomeric (DM) and duplex dimeric (DD) replicative intermediates and individually packaged in AAV capsids. In the nucleus of transduced cells the single-stranded genomes can either be a target for degradation or be converted into transcriptionally active double-stranded templates. The single-to-double strand DNA conversion depends on complementary chain synthesis or on the recruitment of a complementary genome (i.e., intermolecular hybridization). Right panel: According to the same replication model, a rAAV genome with roughly half the size of the wild-type AAV DNA and with one trs mutated, generates DD replicative intermediates with an inverted repeat configuration containing wild-type ITRs at the extremities and mutated ITRs at the axis of symmetry. Single-stranded molecules derived from these DNA structures are packaged in AAV capsids. After uncoating in the target cell nucleus, these molecules can readily fold into double-stranded templates through intramolecular base pairing due to their self-complementary nature (i.e., intramolecular hybridization).
Mentions: Another development in rAAV design is the so-called self-complementary AAV vectors (scAAV) [94]. The scAAV approach builds on the ability of AAV to package replicons with half the size of the wild-type DNA in the form of single-stranded dimeric genomes with an inverted repeat configuration [95]. In the target cell, these self-complementary molecules can readily fold back into double-stranded forms without the need for de novo DNA synthesis or for the annealing of sense and antisense strands (Fig. 6). Ultimately, regardless of the mechanism(s) at play, scAAV lead to enhanced formation of transcription-competent double-stranded genomes thus improving the expression kinetics and yields of vector-encoded products. This scAAV method was subsequently perfected by mutagenesis of one of the two trs sequences to force the generation of dimeric over monomeric replicative forms (Fig. 6) [96]. The main disadvantage of this approach is the need to limit the size of the transgenes that can be delivered to approximately half the length of the already small AAV genome. It is conceivable that this drawback can be tackled by coupling scAAV with heterodimerization strategies. Alternatively, long double-stranded rAAV genomes can be transferred into target cells via capsids of larger viruses such as Ad [97-100], baculovirus [101] or HSV [102]. In some of these hybrid viral vector systems, integration of the rAAV DNA into the AAVS1 locus on human chromosome 19 was accomplished by transient expression of AAV Rep activities in the target cells [38]. Targeted DNA integration is advantageous since it dispels the insertional oncogenesis concerns discussed above.

Bottom Line: The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology.In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed.In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Therapy Section, Department of Molecular Cell Biology, Leiden University Medical Center, the Netherlands. m.goncalves@lumc.nl

ABSTRACT
The initial discovery of adeno-associated virus (AAV) mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV) vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

Show MeSH
Related in: MedlinePlus