DNA-Binding Activity of Adeno-Associated Virus Rep Is Required for Inverted Terminal Repeat-Dependent Complex Formation with Herpes Simplex Virus ICP8

Martín, Alex; Weger, Stefan; Mietzsch, Mario; Slanina, Heiko; Cathomen, Toni; Heilbronn, Regine

doi:10.1128/jvi.06364-11

“…The Rep hybrids that allowed the best packaging efficiency contained the ∼240 N-terminal amino acids of either AAV1 or AAV8 Rep78/68, which contains the DNA-binding and endonuclease domain of the Rep proteins ( 16 ). This region is indispensable for genome replication ( 19 , 40 , 41 ). Thus, it is possible that the Rep hybrids replicate the vector genomes to higher copy numbers prior to their packaging.…”

Section: Resultsmentioning

confidence: 99%

Improved Genome Packaging Efficiency of Adeno-associated Virus Vectors Using Rep Hybrids

Mietzsch

¹

,

Eddington

²

,

Jose

³

et al. 2021

J Virol

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Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2- rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ∼2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. Importance A major byproduct of all Adeno-associated virus (AAV) vector production systems are “empty” capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

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“…The Rep hybrids that allowed the best packaging efficiency contained the ~240 N-terminal amino acids of either AAV1 or AAV8 Rep78/68 which contains the DNA-binding and endonuclease domain of the Rep proteins (16). This region is indispensable for genome replication (18, 37, 38). Thus, it is possible that the Rep hybrids replicate the vector genomes to higher copy numbers prior to their packaging.…”

Section: Resultsmentioning

confidence: 99%

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Mietzsch

¹

,

Eddington

²

,

Jose

³

et al. 2021

Preprint

Self Cite

0

View full text Add to dashboard Cite

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production.

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“…It has been shown that the UL5 helicase activity is necessary for efficient AAV2 DNA replication, whereas the primase-activity of UL52 is redundant [ 61 ]. Although not much is known about the detailed function of ICP8 in AAV2 replication, it seems that it is linked to the interaction with AAV2 Rep78, in an AAV2-ss DNA dependent manner [ 58 , 59 , 62 ].…”

Section: Hsv-1 and Ad Helper Virus Proteins Involved In Aav2 Replicatiomentioning

confidence: 99%

Viral and Cellular Components of AAV2 Replication Compartments

Vogel

¹

,

Seyffert²,

Pereira³

et al. 2013

TOVJ

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Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection.

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DNA-Binding Activity of Adeno-Associated Virus Rep Is Required for Inverted Terminal Repeat-Dependent Complex Formation with Herpes Simplex Virus ICP8

Cited by 8 publications

References 37 publications

Improved Genome Packaging Efficiency of Adeno-associated Virus Vectors Using Rep Hybrids

Improved Genome Packaging Efficiency of Adeno-associated Virus Vectors Using Rep Hybrids

Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

Viral and Cellular Components of AAV2 Replication Compartments

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