Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8+ responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

ABSTRACT Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

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Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression

Human Gene Therapy ◽

10.1089/hum.2019.274 ◽

2020 ◽

Vol 31 (3-4) ◽

pp. 151-162 ◽

Cited By ~ 3

Author(s):

Lauriel F. Earley ◽

Laura M. Conatser ◽

Victoria M. Lue ◽

Amanda L. Dobbins ◽

Chengwen Li ◽

...

Keyword(s):

Transgene Expression ◽

Repeat Sequence ◽

Terminal Repeat ◽

Inverted Terminal Repeat ◽

Adeno Associated Virus ◽

Inverted Terminal Repeat Sequence ◽

Virus Serotype ◽

Terminal Repeat Sequence

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Identification of Attenuated Variants of HIV‐1 Circulating Recombinant Form 01_AE That Are Associated with Slow Disease Progression Due to Gross Genetic Alterations in thenef/Long Terminal Repeat Sequences

The Journal of Infectious Diseases ◽

10.1086/430739 ◽

2005 ◽

Vol 192 (1) ◽

pp. 56-61 ◽

Cited By ~ 25

Author(s):

Makiko Kondo ◽

Takako Shima ◽

Masako Nishizawa ◽

Koji Sudo ◽

Shinya Iwamuro ◽

...

Keyword(s):

Long Terminal Repeat ◽

Disease Progression ◽

Genetic Alterations ◽

Terminal Repeat ◽

Recombinant Form ◽

Slow Disease Progression ◽

Repeat Sequences ◽

Hiv 1

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A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging

Gene Therapy ◽

10.1038/s41434-021-00299-x ◽

2021 ◽

Author(s):

Kumi Adachi ◽

Taro Tomono ◽

Hironori Okada ◽

Yusuke Shiozawa ◽

Motoko Yamamoto ◽

...

Keyword(s):

Plasmid Dna ◽

Virus Production ◽

Single Copy ◽

Dna Packaging ◽

Terminal Repeat ◽

Inverted Terminal Repeat ◽

Adeno Associated Virus

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A cis-Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging

Journal of Virology ◽

10.1128/jvi.76.24.12792-12802.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12792-12802 ◽

Cited By ~ 20

Author(s):

Sergei Musatov ◽

Jill Roberts ◽

Donald Pfaff ◽

Michael Kaplitt

Keyword(s):

Virus Replication ◽

Present Report ◽

Current Model ◽

Terminal Repeat ◽

Biological Role ◽

Inverted Terminal Repeat ◽

Adeno Associated Virus ◽

Cis Acting ◽

Circular Form ◽

Replicative Intermediates

ABSTRACT A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB′), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.

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Inverted Terminal Repeat Sequences Are Important for Intermolecular Recombination and Circularization of Adeno-Associated Virus Genomes

Journal of Virology ◽

10.1128/jvi.79.1.364-379.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 364-379 ◽

Cited By ~ 54

Author(s):

Ziying Yan ◽

Roman Zak ◽

Yulong Zhang ◽

John F. Engelhardt

Keyword(s):

Viral Genome ◽

Terminal Repeat ◽

Inverted Terminal Repeat ◽

Aav Vector ◽

Directional Bias ◽

Adeno Associated Virus ◽

Dual Vector ◽

Vector Systems ◽

Hybrid Genome ◽

Trans Splicing

ABSTRACT The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a β-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute β-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.

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