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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 recombinant AAV split gene principle. An expression unit corresponding to a large gene is roughly divided in two halves. One of them consists of a promoter (solid box with arrowhead), the 5' half of the gene (open box) and a splice donor site (SD) while the other encodes a splice acceptor sequence (SA), the 3' portion of the gene (shaded box) and a polyadenylation signal (solid box). These fragments are independently cloned between two AAV ITRs. Vector stocks are then generated from the resulting shuttle plasmids and are used to co-transduce target cells. Head-to-tail heterodimerization via intermolecular recombination between the two vector DNA molecules restores the full-length expression unit and results in the synthesis of the desired protein after the splicing of the intervening AAV ITR sequences from the primary transcript.
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Figure 5: Diagram of the recombinant AAV split gene principle. An expression unit corresponding to a large gene is roughly divided in two halves. One of them consists of a promoter (solid box with arrowhead), the 5' half of the gene (open box) and a splice donor site (SD) while the other encodes a splice acceptor sequence (SA), the 3' portion of the gene (shaded box) and a polyadenylation signal (solid box). These fragments are independently cloned between two AAV ITRs. Vector stocks are then generated from the resulting shuttle plasmids and are used to co-transduce target cells. Head-to-tail heterodimerization via intermolecular recombination between the two vector DNA molecules restores the full-length expression unit and results in the synthesis of the desired protein after the splicing of the intervening AAV ITR sequences from the primary transcript.

Mentions: The small packaging capacity of AAV particles (about 4.7 kb) [90] is considered one of the main limitations of rAAV vectors since it excludes therapeutically important coding sequences (e.g., dystrophin cDNA) and potent regulatory elements (e.g., albumin promoter). As discussed above, incoming linear rAAV genomes can form concatamers in target cells through intermolecular recombination at their free ends. This phenomenon has been successfully exploited to assemble in target cells large genetic messages through the joining of two independently transduced rAAV genomes each of which encompassing a portion of a large transcriptional unit. mRNA molecules encoding a functional protein are generated from the rAAV DNA head-to-tail heterodimers by splicing out the AAV ITR sequences from the primary transcripts (Fig. 5) [91]. Although this split gene strategy allows expression of almost double-sized transgenes after rAAV-mediated gene delivery, its efficiency is consistently lower than that observed with a single control vector encoding the full-length transgene. Both vectors have to transduce the same cell and only heteroconcatamers with a head-to-tail organization will give rise to a functional full-length gene product. In addition, there are risks associated with the integration into host chromosomes of vectors encoding exclusively regulatory elements or truncated gene products. New work, however, suggests that some of these limitations and concerns can, at least partially, be addressed [92,93].


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

Gonçalves MA - Virol. J. (2005)

Diagram of the recombinant AAV split gene principle. An expression unit corresponding to a large gene is roughly divided in two halves. One of them consists of a promoter (solid box with arrowhead), the 5' half of the gene (open box) and a splice donor site (SD) while the other encodes a splice acceptor sequence (SA), the 3' portion of the gene (shaded box) and a polyadenylation signal (solid box). These fragments are independently cloned between two AAV ITRs. Vector stocks are then generated from the resulting shuttle plasmids and are used to co-transduce target cells. Head-to-tail heterodimerization via intermolecular recombination between the two vector DNA molecules restores the full-length expression unit and results in the synthesis of the desired protein after the splicing of the intervening AAV ITR sequences from the primary transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Diagram of the recombinant AAV split gene principle. An expression unit corresponding to a large gene is roughly divided in two halves. One of them consists of a promoter (solid box with arrowhead), the 5' half of the gene (open box) and a splice donor site (SD) while the other encodes a splice acceptor sequence (SA), the 3' portion of the gene (shaded box) and a polyadenylation signal (solid box). These fragments are independently cloned between two AAV ITRs. Vector stocks are then generated from the resulting shuttle plasmids and are used to co-transduce target cells. Head-to-tail heterodimerization via intermolecular recombination between the two vector DNA molecules restores the full-length expression unit and results in the synthesis of the desired protein after the splicing of the intervening AAV ITR sequences from the primary transcript.
Mentions: The small packaging capacity of AAV particles (about 4.7 kb) [90] is considered one of the main limitations of rAAV vectors since it excludes therapeutically important coding sequences (e.g., dystrophin cDNA) and potent regulatory elements (e.g., albumin promoter). As discussed above, incoming linear rAAV genomes can form concatamers in target cells through intermolecular recombination at their free ends. This phenomenon has been successfully exploited to assemble in target cells large genetic messages through the joining of two independently transduced rAAV genomes each of which encompassing a portion of a large transcriptional unit. mRNA molecules encoding a functional protein are generated from the rAAV DNA head-to-tail heterodimers by splicing out the AAV ITR sequences from the primary transcripts (Fig. 5) [91]. Although this split gene strategy allows expression of almost double-sized transgenes after rAAV-mediated gene delivery, its efficiency is consistently lower than that observed with a single control vector encoding the full-length transgene. Both vectors have to transduce the same cell and only heteroconcatamers with a head-to-tail organization will give rise to a functional full-length gene product. In addition, there are risks associated with the integration into host chromosomes of vectors encoding exclusively regulatory elements or truncated gene products. New work, however, suggests that some of these limitations and concerns can, at least partially, be addressed [92,93].

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