Limits...
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.

Show MeSH

Related in: MedlinePlus

Overview of the initial recombinant AAV production system. The generation of the first infectious clones of AAV permitted functional dissection of the virus genome. This allowed the construction of plasmids encoding rAAV genomes in which the minimal complement of wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a gene of interest (transgene) instead of the AAV rep and cap genes. When these constructs are transfected into packaging cells together with a rep and cap expression plasmid they lead to the production of rAAV particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle were originally introduced by infection of the packaging cells with wild-type Ad as depicted. Current transfection-based production methods make use of recombinant DNA encoding the helper activities instead of Ad infection. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to the accumulation of replicative intermediates both in the duplex monomer (DM) and duplex dimer (DD) forms. A fraction of this de novo synthesized DNA is incorporated in the single-stranded format into preformed empty capsids most likely through the catalytic activities of the Rep52 and Rep40 proteins. The resulting infectious rAAV virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation allows the selective elimination of the helper virus from the final rAAV preparation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1131931&req=5

Figure 4: Overview of the initial recombinant AAV production system. The generation of the first infectious clones of AAV permitted functional dissection of the virus genome. This allowed the construction of plasmids encoding rAAV genomes in which the minimal complement of wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a gene of interest (transgene) instead of the AAV rep and cap genes. When these constructs are transfected into packaging cells together with a rep and cap expression plasmid they lead to the production of rAAV particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle were originally introduced by infection of the packaging cells with wild-type Ad as depicted. Current transfection-based production methods make use of recombinant DNA encoding the helper activities instead of Ad infection. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to the accumulation of replicative intermediates both in the duplex monomer (DM) and duplex dimer (DD) forms. A fraction of this de novo synthesized DNA is incorporated in the single-stranded format into preformed empty capsids most likely through the catalytic activities of the Rep52 and Rep40 proteins. The resulting infectious rAAV virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation allows the selective elimination of the helper virus from the final rAAV preparation.

Mentions: Historically, most recombinant AAV (rAAV) vectors have been based on serotype 2 (AAV2) that constitutes the prototype of the genus [51,52]. Important to those pursuing the use of rAAV for gene therapy applications is the defectiveness of the parental virus and its presumed non-pathogenic nature. The realization that a molecularly cloned AAV genome could in Ad-infected cells recapitulate the lytic phase of the AAV life cycle and give rise to infectious virions enabled not only the detailed genetic analyses of the virus but provided, in addition, a substrate to generate rAAV particles [53]. The latter task was facilitated by the fact that the AAV ITRs contain all cis-acting elements involved in genome rescue, replication and packaging. Furthermore, since the AAV ITRs are segregated from the viral encoding regions, rAAV design can follow the whole-gene-removal or "gutless" vector rational of, for instance, retrovirus-based vectors in the sense that the cis-acting elements involved in genome amplification and packaging are in linkage with the heterologous sequences of interest, whereas the virus encoding sequences necessary for genome replication and virion assembly are provided in trans (Fig. 4). Typically, rAAV particles are generated by transfecting producer cells with a plasmid containing a cloned rAAV genome composed of foreign DNA flanked by the 145 nucleotide-long AAV ITRs and a construct expressing in trans the viral rep and cap genes. In the presence of Ad helper functions, the rAAV genome is subjected to the wild-type AAV lytic processes by being rescued from the plasmid backbone, replicated and packaged into pre-formed AAV capsids as single-stranded molecules.


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

Gonçalves MA - Virol. J. (2005)

Overview of the initial recombinant AAV production system. The generation of the first infectious clones of AAV permitted functional dissection of the virus genome. This allowed the construction of plasmids encoding rAAV genomes in which the minimal complement of wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a gene of interest (transgene) instead of the AAV rep and cap genes. When these constructs are transfected into packaging cells together with a rep and cap expression plasmid they lead to the production of rAAV particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle were originally introduced by infection of the packaging cells with wild-type Ad as depicted. Current transfection-based production methods make use of recombinant DNA encoding the helper activities instead of Ad infection. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to the accumulation of replicative intermediates both in the duplex monomer (DM) and duplex dimer (DD) forms. A fraction of this de novo synthesized DNA is incorporated in the single-stranded format into preformed empty capsids most likely through the catalytic activities of the Rep52 and Rep40 proteins. The resulting infectious rAAV virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation allows the selective elimination of the helper virus from the final rAAV preparation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Overview of the initial recombinant AAV production system. The generation of the first infectious clones of AAV permitted functional dissection of the virus genome. This allowed the construction of plasmids encoding rAAV genomes in which the minimal complement of wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a gene of interest (transgene) instead of the AAV rep and cap genes. When these constructs are transfected into packaging cells together with a rep and cap expression plasmid they lead to the production of rAAV particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle were originally introduced by infection of the packaging cells with wild-type Ad as depicted. Current transfection-based production methods make use of recombinant DNA encoding the helper activities instead of Ad infection. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to the accumulation of replicative intermediates both in the duplex monomer (DM) and duplex dimer (DD) forms. A fraction of this de novo synthesized DNA is incorporated in the single-stranded format into preformed empty capsids most likely through the catalytic activities of the Rep52 and Rep40 proteins. The resulting infectious rAAV virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation allows the selective elimination of the helper virus from the final rAAV preparation.
Mentions: Historically, most recombinant AAV (rAAV) vectors have been based on serotype 2 (AAV2) that constitutes the prototype of the genus [51,52]. Important to those pursuing the use of rAAV for gene therapy applications is the defectiveness of the parental virus and its presumed non-pathogenic nature. The realization that a molecularly cloned AAV genome could in Ad-infected cells recapitulate the lytic phase of the AAV life cycle and give rise to infectious virions enabled not only the detailed genetic analyses of the virus but provided, in addition, a substrate to generate rAAV particles [53]. The latter task was facilitated by the fact that the AAV ITRs contain all cis-acting elements involved in genome rescue, replication and packaging. Furthermore, since the AAV ITRs are segregated from the viral encoding regions, rAAV design can follow the whole-gene-removal or "gutless" vector rational of, for instance, retrovirus-based vectors in the sense that the cis-acting elements involved in genome amplification and packaging are in linkage with the heterologous sequences of interest, whereas the virus encoding sequences necessary for genome replication and virion assembly are provided in trans (Fig. 4). Typically, rAAV particles are generated by transfecting producer cells with a plasmid containing a cloned rAAV genome composed of foreign DNA flanked by the 145 nucleotide-long AAV ITRs and a construct expressing in trans the viral rep and cap genes. In the presence of Ad helper functions, the rAAV genome is subjected to the wild-type AAV lytic processes by being rescued from the plasmid backbone, replicated and packaged into pre-formed AAV capsids as single-stranded molecules.

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