Dataset Information

OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

ABSTRACT: Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates. OneBac 2.0 represents a 2-fold advancement: First, enhanced VP1 proportions in AAV5 capsids lead to vastly increased per-particle infectivity rates. Second, collateral packaging of foreign DNA is suppressed by removal of the Rep-binding element (RBE). In this study we show that this advancement of AAV5 packaging can be translated to OneBac 2.0-derived packaging systems for alternative AAV serotypes. By removal of the RBE, collateral packaging of nonvector DNA was drastically reduced in all newly tested serotypes (AAV1, AAV2, and AAV8). However, the splicing-based strategy to enhance VP1 expression in order to increase AAV5 infectivity hardly improved infectivity rates of AAV-1, -2, or -8 compared with the original OneBac cell lines. Our results emphasize that OneBac 2.0 represents an advancement for scalable, high-titer production of various AAV serotypes, leading to AAV particles with minimal packaging of foreign DNA.

SUBMITTER: Mietzsch M

PROVIDER: S-EPMC5586161 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

Mietzsch Mario M Hering Henrik H Hammer Eva-Maria EM Agbandje-McKenna Mavis M Zolotukhin Sergei S Heilbronn Regine R

Human gene therapy methods 20170201 1

Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high ...[more]

PMID: 28125901

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Project description:Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Dataset Information

OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

Publications

OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

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