Marker rescue of adeno-associated virus (AAV) capsid mutants: a novel approach for chimeric AAV production

Abstract

Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact rep gene, and a mutated cap gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus dl309 helper virus. However, the mutation in the cap gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The cap genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3 cap gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric cap genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.

FIG. 1.

Overview of the experimental system. (A) Capsid mutants used in the present study. An expanded region of the AAV2 capsid DNA sequence (nt 3760 to 3882) and VP1 protein sequence (amino acids 521 to 561) is shown. The details of the construction of the 1n532 and 1n562 capsid mutants were described in Materials and Methods. The location of the 12-bp linker insertion found in each of the mutants is in italicized. In addition to containing the 12-bp linker, the 1n532 mutant lacks 5 nt (underlined), whereas the 1n562 mutant contains an additional 5 nt (underlined). As a result, the 1n532 and 1n562 capsid mutants truncate at VP1 amino acid positions 532 and 562, respectively, and do not assemble viral particles. The H/N3761 mutant has been previously described (31) and contains a 12-bp in-frame linker (encoding the amino acids AISP) inserted at AAV2 nt 3761. The H/N3761 mutant assembles particles but is noninfectious due to a lack of heparin binding. (B) Marker rescue strategy to generate chimeric AAV. DNA from the three mutants (H/N3761, 1n532, and 1n562) was digested with PvuII and cotransfected with the AAV3 PCR product. After the transfection, the 293 cells were coinfected with adenovirus dl309. The lysates from the transfections (T) were applied in two successive cycles (C1 and C2) to HeLa cells and then infected with adenovirus dl309. The resulting viruses were analyzed by dot blot, Western blot, PCR, and DNA sequencing analyses.

FIG. 2.

(A) Dot blot hybridization of the H/N3671, 1n532, and 1n562 mutants. 293 cells were transfected (T) with each of the mutant plasmids (H/N3761, 1n562, or 1n532) and with (+AAV3) or without (−AAV3) AAV3 capsid DNA fragments and then infected with dl309. The lysate from each was subjected to two rounds of cycling onto HeLa cells (C1 and C2). Then, 10 μl of freeze-thaw lysates from the transfection (T), cycle 1 (C1), or cycle 2 (C2) of H/N3761, 1n532, or 1n562 were applied to GeneScreen Plus membrane and probed with the psub201(+) plasmid, which had been random prime labeled. (B) Analyses of the capsid protein composition from cellular lysates. Protein lysates from the transfection of H/N3761 (lane 1), 1n532 (lane 2), and 1n562 (lane 3); the cotransfection of AAV3 capsid DNA fragments with H/N3761 (lane 4), 1n532 (lane 5), and 1n562 (lane 6); or lysates from each of the above transfections which had been cycled twice (C2) onto HeLa cells (lanes 7 to 12) were resolved on a 10% polyacrylamide electrophoresis gel and transblotted to a Hybond ECL membrane. The amount of total protein was determined by BCA assay, and 5 μg of total protein was loaded per lane. The membrane was probed with monoclonal antibody B1 directed against the carboxyl termini of the AAV2 VP3 capsid protein (45). The locations of VP1, VP2, and VP3 capsid proteins are indicated.

FIG. 3.

PCR analyses of rescued virus from independent marker rescue experiments. (Top) PCR analyses were performed on two independent samples from H/N3761 (lanes 1, 2, 7, and 8), three independent samples from 1n562 (lanes 3 to 6, 9, and 10), one sample from the 1n532 marker rescue experiments (lanes 11 and 12), AAV2 plasmid (lanes 13 and 14), and AAV3 plasmid (lanes 15 and 16). Lysates from the second cycle (C2) on HeLa cells from each of these experiments were amplified with either AAV2-specific primers (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or AAV3-specific primers (lanes 2, 4, 6, 8, 10, 12, 14, and 16). The location of the 2.2-kb PCR product is indicated by an arrow. (Bottom) Diagram of the pxr2AN shuttle vector. This shuttle vector is a modification of the pxr2 vector generated by Rabinowitz and Samulski (30), except that AflII and NotI sites flanking the capsid regions were engineered to facilitate cloning of PCR products.

FIG. 4.

Four classes of AAV2/AAV3 chimeras are produced from marker rescue experiments. The four classes of AAV2/AAV3 capsid chimeras identified are depicted, and arrows indicate the approximate sites where homologous recombination occurred between the capsid DNA of serotypes 2 and 3. Whether these classes were chimeras at the amino acid level is indicated next to each class. Representative AAV helper clones from each class were all able to generate rAAV in a standard triple-transfection assay.

FIG. 5.

Sequence alignment of class III chimera clone 6q. Sequence of a portion of the AAV2, AAV3, and AAV2/3 chimeric clone 6q (corresponding to AAV3 capsid open reading frame nt 1538 to 1605) is shown. A 16-bp region of AAV3 (underlined; nt 1564 to 1579, based on the AAV3 VP1) was identified as the minimal AAV3 region necessary to rescue the defects of the AAV2 virus (the line above indicates the location of the mutated region). For the generation of the chimeric clone 6q, an 11-bp region on the 5′ end (bp 1550 to 1560) and a 20-bp region on the 3′ end of the AAV3 (bp 1580 to 1599) were identified as crossover sites. The sites where homologous recombination is presumed to occur are shown in boxes.

FIG. 6.

Predicted utility of the marker rescue approach to generate chimeric AAV vectors. The marker rescue approach can be utilized to generate chimeric virus from any combination of AAV serotypes. A capsid mutant of any serotype serves as the marker rescue plasmid template. (A) The mutation can be rescued via cotransfection capsid DNA from any other template. (B) After transfection of the cells with the mutant plasmid and capsid DNA, a mixed population of viral genomes is produced that can be PCR amplified and cloned into the shuttle vector, and individual clones can then be assessed for biological properties. (C and D) Alternatively, as a selection step the transfection lysate can be cycled onto any cell type of interest (C) or can be used to directly infect animals (D). (E) Ultimately, the cycling onto cells or in animals serves to enrich for a predominant viral recombinant.