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Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

ABSTRACT: The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 components in HEK 293 cells. The Rep 78 and ZFN-based approaches were also compared in HEK 293 cells and in human induced pluripotent stem cells using integrase deficient lentiviral vectors. The targeting efficiencies involving the Rep 78 protein were similar to those involving the AAVS1-specific ZFN, while the targeting specificity for the Rep 78 protein was lower compared to that of the ZFN. It is anticipated that the Rep 78 nickase-based targeting approach may ultimately contribute to the reduction of risks associated with other genome editing approaches involving DNA double-strand breaks.

SUBMITTER: Li P

PROVIDER: S-EPMC6016731 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

Li Pingjuan P Marino Michael P MP Zou Jizhong J Argaw Takele T Morreale Michael T MT Iaffaldano Brian J BJ Reiser Jakob J

Human gene therapy methods 20180601 3

The adeno-associated virus serotype 2 (AAV2) Rep 78 protein, a strand-specific endonuclease (nickase) promotes site-specific integration of transgene sequences bearing homology arms corresponding to the AAVS1 safe harbor locus. To investigate the efficiency and specificity of this approach, plasmid-based donor vectors were tested in concert with nuclease encoding vectors, including an engineered version of the AAV2 Rep 78 protein, an AAVS1-specific zinc finger nuclease (ZFN), and the CRISPR-Cas9 ...[more]

PMID: 29860898

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Project description:Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 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 single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product 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 approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product 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.

Project description:Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T?Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S?A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (~9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Dataset Information

Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

Publications

Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein.

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