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Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors.

ABSTRACT: Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.

SUBMITTER: Ling C

PROVIDER: S-EPMC4856060 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors.

Ling Chen C Yin Zifei Z Li Jun J Zhang Daniel D Aslanidi George G Srivastava Arun A

Molecular therapy. Methods & clinical development 20160504

Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in ...[more]

PMID: 27200382

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Project description:Adeno-associated virus serotype 5 (AAV5) is being developed as a gene delivery vector for several diseases, including hemophilia and Huntington's disease, and has a demonstrated efficient transduction in liver, lung, skeletal muscle, and the central nervous system. One limitation of AAV gene delivery is preexisting neutralizing antibodies, which present a significant challenge for vector effectiveness in therapeutic applications. Here, we report the cryo-electron microscopy (cryo-EM) and image-reconstructed structure of AAV5 in complex with a newly generated monoclonal antibody, HL2476, at 3.1-Å resolution. Unlike other available anti-AAV5 capsid antibodies, ADK5a and ADK5b, with epitopes surrounding the 5-fold channel of the capsid, HL2476 binds to the 3-fold protrusions. To elucidate the capsid-antibody interactions, the heavy and light chains were sequenced and their coordinates, along with the AAV5 viral protein, assigned to the density map. The high resolution of the complex enabled the identification of interacting residues at the 3-fold protrusions of the capsid, including R483, which forms two hydrogen bonds with the light chain of HL2476. A panel of AAV5 variants was generated and analyzed by native dot immunoblot and transduction assays. This identified variants with antibody escape phenotypes that maintain infectivity.IMPORTANCE Biologics based on recombinant AAVs (rAAVs) are increasingly becoming attractive human gene delivery vehicles, especially after the approval of Glybera in Europe and Luxturna in the United States. However, preexisting neutralizing antibodies against the AAV capsids in a large percentage of the human population limit wide-spread utilization of these vectors. To circumvent this problem, stealth vectors must be generated that are undetectable by these antibodies. This study details the high-resolution characterization of a new antigenic region on AAV5, a vector being developed for numerous delivery applications. The structure of AAV5 complexed with HL2476, a novel antibody, was determined by cryo-EM to 3.1-Å resolution. The resolution of the density map enabled the identification of interacting residues between capsid and antibody and the determinants of neutralization. Thus, the information obtained from this study can facilitate the generation of host immune escape vectors.

Dataset Information

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors.

Publications

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors.

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