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DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

ABSTRACT: Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

SUBMITTER: Koerber JT

PROVIDER: S-EPMC2683895 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

Koerber James T JT Jang Jae-Hyung JH Schaffer David V DV

Molecular therapy : the journal of the American Society of Gene Therapy 20080826 10

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to ...[more]

PMID: 18728640

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Project description:The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA(+) domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle.The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

Dataset Information

DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

Publications

DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

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